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Materials Science
Computer Science
Medicine

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Chemistry
Materials Science

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Nanomaterials for Electrochemical Immunosensing

Electrochemical immunosensors resulting from a combination of the traditional immunoassay approach with modern biosensors and electrochemical analysis constitute a current research hotspot. They exhibit both the high selectivity characteristics of immunoassays and the high sensitivity of electrochemical analysis, along with other merits such as small volume, convenience, low cost, simple preparation, and real-time on-line detection, and have been widely used in the fields of environmental monitoring, medical clinical trials and food analysis. Notably, the rapid development of nanotechnology and the wide application of nanomaterials have provided new opportunities for the development of high-performance electrochemical immunosensors. Various nanomaterials with different properties can effectively solve issues such as the immobilization of biological recognition molecules, enrichment and concentration of trace analytes, and signal detection and amplification to further enhance the stability and sensitivity of the electrochemical immunoassay procedure. This review introduces the working principles and development of electrochemical immunosensors based on different signals, along with new achievements and progress related to electrochemical immunosensors in various fields. The importance of various types of nanomaterials for improving the performance of electrochemical immunosensor is also reviewed to provide a theoretical basis and guidance for the further development and application of nanomaterials in electrochemical immunosensors.

Introduction
Biosensors are a special kind of devices with specific recognition ability, which usually employ specific biological macromolecules such as enzymes, receptors, or antibodies as recognition elements [1]. Owing to their specific recognition or rapid catalysis of the recognition elements, biosensors can meet the requirements of specificity and rapidity, as well as the real-time and on-line detection needs, of the modern analysis process and thus fill a wider research and development space [2]. Immunosensors, an important sub-branch of biosensors, function based on the specific recognition between antigens and antibodies [3,4]. Theoretically, all biological macromolecules such as proteins, microbes that can be used as antigens and other compounds, including hormones, antibiotics, persistent environmental pollutants and toxins that can be used as haptens can induce the production of antibodies with specific recognition ability. This feature represents a powerful driving force for the further development of immunosensors, which accordingly represent some of the most in-depth-studied biosensors. In recent years, studies on immunosensors have mainly focused on how to accurately quantify the signal response with respect to the antigen-antibody binding and signal conversion/amplification, which has led to remarkable achievements in the respective fields [5][6][7]. Based on their different forms of signal

Amperometric Immunosensors
Amperometric immunosensors represent a highly developed biosensing field that has led to the appearance of several commercialized products [24]. This kind of sensors can measure the current change at a constant voltage signifying the occurrence of a redox reaction on the analyte, because the resulting current on the sensing electrode surface is proportional to the analyte concentration [25]. This type of response system is highly sensitive and highly linearly related to the analyte concentration, allowing relatively straight forward data processing and conversion, thus making it ideal for immunochemical sensing. Following the first report from Aizawa et al. [26] in 1979 on an amperometric immunosensor for human chorionic gonadotropin, this type of sensor has attracted much research attention and developed rapidly. Owing to the non-electrochemical activity of the antigen and antibody biomolecules, indirect electrochemical immunoassays are mainly used for measuring the content of immune protein molecules. Specifically, by labeling an electroactive substance or enzyme on an antigen or antibody, the concentration of the antigen or antibody in the sample can be indirectly measured by monitoring the current change caused the reaction of the substrate of the electroactive substance or the enzyme catalyzed reaction between the immobilized biomolecule and the biomolecule in the solution. In this mode, the resulting current signal of the antigen antibody binding reaction has been amplified using the markers such as enzymes (alkaline phosphatase [27,28], horseradish peroxidase (HRP) [29,30] or glucose oxidase [31,32]) or nanomaterials (Au nanoparticles (AuNPs) [33], graphene [34], carbon nanotubes (CNTs) [35,36] or silica nanoparticles [37,38]), which has allowed the detection sensitivity and reproducibility to be increased to a certain extent. Additionally, according to the enzyme-linked immunoassay (ELISA) principle, this kind of immunosensors can be further divided into two categories based on competition or sandwich methodologies ( Figure 1) [39]. substance or enzyme on an antigen or antibody, the concentration of the antigen or antibody in the sample can be indirectly measured by monitoring the current change caused the reaction of the substrate of the electroactive substance or the enzyme catalyzed reaction between the immobilized biomolecule and the biomolecule in the solution. In this mode, the resulting current signal of the antigen antibody binding reaction has been amplified using the markers such as enzymes (alkaline phosphatase [27,28], horseradish peroxidase (HRP) [29,30] or glucose oxidase [31,32]) or nanomaterials (Au nanoparticles (AuNPs) [33], graphene [34], carbon nanotubes (CNTs) [35,36] or silica nanoparticles [37,38]), which has allowed the detection sensitivity and reproducibility to be increased to a certain extent. Additionally, according to the enzyme-linked immunoassay (ELISA) principle, this kind of immunosensors can be further divided into two categories based on competition or sandwich methodologies ( Figure 1) [39]. Doldán et al. [40] fabricated a sandwich mode amperometric immunosensor by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for the detection of captured antigen ( Figure 2). Signal amplification is obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for determination. Figure 2. Schematic diagram of amperometric immunosensor using sandwich mode by immobilizing rabbit antihuman CD9 antibodies on gold substrates. Reproduced with permission from reference [40]. Copyright 2016 American Chemical Society. Doldán et al. [40] fabricated a sandwich mode amperometric immunosensor by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for the detection of captured antigen ( Figure 2). Signal amplification is obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for determination. substance or enzyme on an antigen or antibody, the concentration of the antigen or antibody in the sample can be indirectly measured by monitoring the current change caused the reaction of the substrate of the electroactive substance or the enzyme catalyzed reaction between the immobilized biomolecule and the biomolecule in the solution. In this mode, the resulting current signal of the antigen antibody binding reaction has been amplified using the markers such as enzymes (alkaline phosphatase [27,28], horseradish peroxidase (HRP) [29,30] or glucose oxidase [31,32]) or nanomaterials (Au nanoparticles (AuNPs) [33], graphene [34], carbon nanotubes (CNTs) [35,36] or silica nanoparticles [37,38]), which has allowed the detection sensitivity and reproducibility to be increased to a certain extent. Additionally, according to the enzyme-linked immunoassay (ELISA) principle, this kind of immunosensors can be further divided into two categories based on competition or sandwich methodologies ( Figure 1) [39]. Doldán et al. [40] fabricated a sandwich mode amperometric immunosensor by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for the detection of captured antigen ( Figure 2). Signal amplification is obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for determination. Figure 2. Schematic diagram of amperometric immunosensor using sandwich mode by immobilizing rabbit antihuman CD9 antibodies on gold substrates. Reproduced with permission from reference [40]. Copyright 2016 American Chemical Society. A competitive disposable amperometric immunosensor was firstly developed by Manfredi et al. [41] based on gliadin-functionalized carbon/nanogold screen-printed electrodes for rapid determination of celiotoxic prolamins. The developed immunocompetitive assay achieved good sensitivity for gliadin in ethanol extracts, giving limit of detection and limit of quantitation at 8 and 22 ng·mL −1 , respectively.

Potentiometric Immunosensors
Potentiometric immunosensors comprise a kind of biosensing device that performs immunoassays by measuring the change of electrochemical potential for direct or indirect detection of various antigens with the characteristics of real-time monitoring and rapid response time. In 1975, Janata [42] first reported the change of membrane potential caused by an antigen binding to an antibody immobilized on the surface of a metal electrode using polyvinyl chloride film. When the antibody or antigen was detected by the sensitive membrane of the sensor, the membrane potential (or the electrode potential) could be changed, which has a logarithmic relationship between the change of potential and antigen or antibody concentration.
Based on the different principle of membrane-and ion-electrode potential measurement, two kinds of potentiometric immunosensors have been developed. The measurement of membrane potential is susceptible to nonspecific adsorption issues, resulting in a large background current and low detection sensitivity, limiting its further practical application. In contrast, the high sensitivity of an immunoassay is combined with the high selectivity of an ion-selective electrode in the ion selective electrode immunosensor which can monitor the change of electrode potential caused by an antibody-immobilized ionic carrier on the surface of the electrode because of the specific binding of the antigen, further reflecting the concentration of the antigen. For example, an ion-selective immunosensor for the determination of insulin has been constructed by Ghindilis et al [43], in which using lactase labeled antibody for insulin and according that utilizes a lactase-labeled antibody for insulin. According to the catalysis of lactase oxidation, the resulting potential changes on the electrode could be recorded to achieve insulin monitoring. The introduction of nanoparticles provides new means for improving the performance of ion-selective potential immunosensors, especially in terms of increasing the linear response range and enhancing the sensitivity. Fu et al. [44] have adsorbed AuNPs to the surface of the electrode by self-assembly of mercaptoethylamine and then immobilizing the immunoglobulin antibody (anti-IgG) to develop a highly sensitive potentiometric immunosensor. The performance of the AuNPs-introduced sensing device was confirmed by cyclic voltammetry and electrochemical impedance techniques to show a detection limit of 12 ng·mL −1 , which is similar to that of the ELISA method. Tang et al. [45] have constructed a similar immunosensor on the surface of a glassy carbon electrode using AuNPs for anti-diphtheria immobilization for the detection of diphtheria toxoid in biological samples. The linear response range and detection limit achieved were 2.4-60.0 ng·mL −1 and 5.2 ng·mL −1 , respectively. Notably, these studies also demonstrate that the nanomaterials can effectively solve the shortcomings of low sensitivity and narrow linear range of potentiometric immunosensors.

Impedance Immunosensor
Electrochemical impedance spectroscopy is an effective method to study the properties of conductive materials and interfaces, and has been widely used in the field of electrochemical sensors [46]. For a sensor with impedance characteristics, the combination of its capacitance, inductance, and resistance characteristics can produce a specific impedance signal. Therefore, when the surrounding environment of the sensor is modified to cause a change of the phenomenon underlies the response of sensors based on electrochemical impedance. Impedance immunosensors can real-time monitor the change on the electron transfer impedance in solutions before and after an immune reaction to achieve assessment of the tested analyte. Compared with other analytical methods, this detection mode is free of the markers, affords less damage to the immune system, and involves relatively simple operation and sensor preparation processes [47].
During the fabrication of impedance sensors, nanomaterials are the most commonly used for antibody immobilization, which not only increases the amount of antibody loaded but also maintains antibody activity over a longer period of time to meet practical needs. For example, Chen et al. [48] reported a simple preparation process of one enzyme-free labeled impedance immunosensor based on 3D-orderedmacroporousgoldfilm. The film was employed to immobilize the antibody for reaction with the corresponding antigen to form immune complexes, which could inhibit the electrical activity of the Fe(CN) 6 3−/4− probe and further achieve the sensitive detection of C-reactive protein.
To construct a novel impedance immunosensor, Li et al. [49] prepared a novel ionic-liquid-doped polyaniline inverse opals via electropolymerization method on an electrode surface, wherein AuNPs self-assembly was utilized for antibody immobilization. Yuan et al. [50] developed a sandwich impedance immunoassay using the complex of CNTs and AuNPs as electrode matrix for antibody immobilization. As the capture of silicon nanosphere markers on the electrode surface is not conducive to the transfer of electrons, the electrical activity of the Fe(CN) 6 3−/4− probe was thereby inhibited, achieving the electrochemical determination of the target glycoprotein ( Figure 3). During the fabrication of impedance sensors, nanomaterials are the most commonly used for antibody immobilization, which not only increases the amount of antibody loaded but also maintains antibody activity over a longer period of time to meet practical needs. For example, Chen et al. [48] reported a simple preparation process of one enzyme-free labeled impedance immunosensor based on 3D-orderedmacroporousgoldfilm. The film was employed to immobilize the antibody for reaction with the corresponding antigen to form immune complexes, which could inhibit the electrical activity of the Fe(CN)6 3−/4− probe and further achieve the sensitive detection of C-reactive protein. To construct a novel impedance immunosensor, Li et al. [49] prepared a novel ionic-liquid-doped polyaniline inverse opals via electropolymerization method on an electrode surface, wherein AuNPs selfassembly was utilized for antibody immobilization. Yuan et al. [50] developed a sandwich impedance immunoassay using the complex of CNTs and AuNPs as electrode matrix for antibody immobilization. As the capture of silicon nanosphere markers on the electrode surface is not conducive to the transfer of electrons, the electrical activity of the Fe(CN)6 3−/4− probe was thereby inhibited, achieving the electrochemical determination of the target glycoprotein ( Figure 3). Impedance sensors with advantages of ease of generation, low-cost, and rapid response also represent good prospects in the field of microbial analysis [51]. For example, Yang et al. [52] has constructed a microelectrode impedance sensor without marker to detect Escherichia coli O157:H7 in food samples. The target bacteria was adsorbed on the indium tin oxide (ITO) microelectrode surface and caused an impedance response, which had a proportional relationship with the target bacteria concentration in the range of 4.36 × 10 5 -4.36 × 10 8 cfu/mL. Nevertheless, the detection limit of the impedance sensor is still relatively high compared to that of other methods, and the impedance analysis system for complex samples also needs to be improved, requiring substantial effort to render this technology more practical and popular.

Conductometric Immunosensors
Many chemical reactions are accompanied by the generation and consumption of varieties of plasma that can change the conductivity in solutions, thus allowing the determination of conductivity in a variety of chemical systems. The conductometric immunosensors can detect an antigen by measuring the change of the conductivity caused by the change of the type and number of ions in the solution prior to and following immunization. This measurement is usually carried out by immobilizing a specific enzyme on the metal electrode surface to determine the conductivity under the action of an electric field. Yagiuda et al. [53] has successfully fabricated a novel conductivitybased immunosensor for sensitive detection of methamphetamine in human urine, greatly reducing Impedance sensors with advantages of ease of generation, low-cost, and rapid response also represent good prospects in the field of microbial analysis [51]. For example, Yang et al. [52] has constructed a microelectrode impedance sensor without marker to detect Escherichia coli O157:H7 in food samples. The target bacteria was adsorbed on the indium tin oxide (ITO) microelectrode surface and caused an impedance response, which had a proportional relationship with the target bacteria concentration in the range of 4.36 × 10 5 -4.36 × 10 8 cfu/mL. Nevertheless, the detection limit of the impedance sensor is still relatively high compared to that of other methods, and the impedance analysis system for complex samples also needs to be improved, requiring substantial effort to render this technology more practical and popular.

Conductometric Immunosensors
Many chemical reactions are accompanied by the generation and consumption of varieties of plasma that can change the conductivity in solutions, thus allowing the determination of conductivity in a variety of chemical systems. The conductometric immunosensors can detect an antigen by measuring the change of the conductivity caused by the change of the type and number of ions in the solution prior to and following immunization. This measurement is usually carried out by immobilizing a specific enzyme on the metal electrode surface to determine the conductivity under the action of an electric field. Yagiuda et al. [53] has successfully fabricated a novel conductivity-based immunosensor for sensitive detection of methamphetamine in human urine, greatly reducing the detection cost. Muhammad-Tahir et al. [54] reported a conductometric biosensor for detecting foodborne pathogens (Escherichia coli O157:H7 and Salmonella spp). The developed biosensor consisting of two components: an immunosensor on electrochemical sandwich immunoassay and a reader for signal measurement, provided a specific, sensitive, low volume, and near real-time detection mechanism.
Tang et al. [55] have designed a simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of immobilizing monoclonal AFP primary antibodies on an interdigitated conductometric transducer, while the antibodies for detection were assembled using nanocarbon-conjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP-anti-AFP). A new conductometric immunoassay was also prepared by Xu et al. [56] based on biofunctionalized Prussian blue-gold hybrid nanostructure for sensitive determination of tissue polypeptide antigen ( Figure 4). the detection cost. Muhammad-Tahir et al. [54] reported a conductometric biosensor for detecting foodborne pathogens (Escherichia coli O157:H7 and Salmonella spp). The developed biosensor consisting of two components: an immunosensor on electrochemical sandwich immunoassay and a reader for signal measurement, provided a specific, sensitive, low volume, and near real-time detection mechanism. Tang et al. [55] have designed a simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of immobilizing monoclonal AFP primary antibodies on an interdigitated conductometric transducer, while the antibodies for detection were assembled using nanocarbonconjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP-anti-AFP). A new conductometric immunoassay was also prepared by Xu et al. [56] based on biofunctionalized Prussian blue-gold hybrid nanostructure for sensitive determination of tissue polypeptide antigen ( Figure 4).  Although the conductometric immunosensors have been utilized since the early period of immunosensor technologic development, owing to the large influence of ionic strength in the testing samples, buffer volume and the nonspecific adsorption effect in the measurement process, the development of improved detection and analysis using this type of immunosensor has been relatively delayed.

Capacitive Immunosensors
Electrochemical capacitive immunosensors represent a new type of sensor with remarkable merits of high sensitivity, simple structure, easy integration and no need for labels. In particular, in recent years these have been demonstrated with respect to their capacity to facilitate direct and rapid detection [57]. When the metal electrode is in contact with the electrolyte solution, an electric double layer is generated at the interface between the electrode and the testing solution [58]. The interface capacitance of the testing solution can be sensitive to the physical and chemical properties of the interface changes. Specifically, when the material with low polarity is adsorbed to the electrode surface, the thickness of the interface layer will increase and the interface dielectric constant will be reduced, resulting in the decrease of the interface capacitance. It is well known that the protein molecules represent biomacromolecules with large molecular weight and low polarity. When a protein molecule is adsorbed to the electrode surface, the interface capacitance from the electrode solution is significantly reduced. Thus, in a capacitive immunosensor, the antibody is firstly immobilized on the surface of the electrode, and when the antigen-antibody is bound to the electrode surface, the interface capacitance is correspondingly reduced to reflect the amount of antigen to be Although the conductometric immunosensors have been utilized since the early period of immunosensor technologic development, owing to the large influence of ionic strength in the testing samples, buffer volume and the nonspecific adsorption effect in the measurement process, the development of improved detection and analysis using this type of immunosensor has been relatively delayed.

Capacitive Immunosensors
Electrochemical capacitive immunosensors represent a new type of sensor with remarkable merits of high sensitivity, simple structure, easy integration and no need for labels. In particular, in recent years these have been demonstrated with respect to their capacity to facilitate direct and rapid detection [57]. When the metal electrode is in contact with the electrolyte solution, an electric double layer is generated at the interface between the electrode and the testing solution [58]. The interface capacitance of the testing solution can be sensitive to the physical and chemical properties of the interface changes. Specifically, when the material with low polarity is adsorbed to the electrode surface, the thickness of the interface layer will increase and the interface dielectric constant will be reduced, resulting in the decrease of the interface capacitance. It is well known that the protein molecules represent biomacromolecules with large molecular weight and low polarity. When a protein molecule is adsorbed to the electrode surface, the interface capacitance from the electrode solution is significantly reduced. Thus, in a capacitive immunosensor, the antibody is firstly immobilized on the surface of the electrode, and when the antigen-antibody is bound to the electrode surface, the interface capacitance is correspondingly reduced to reflect the amount of antigen to be detected. For example, Mirsky et al. [59] have compared several methods forthe immobilization of protein molecules on self-assembled monolayers. They determined that by using N-hydroxysuccinimide or carbodiimide to activate the carboxyl groups on the ω-mercapto-hexadecanoic acid monolayer, following albumin immobilization on this monolayer, the sensor surface could still maintain high dielectric properties. Accordingly, a detection limit of human serum albumin of 15 × 10 −8 mol·L −1 was achieved using a sandwich capacitive immunosensor.
In general, various types of electrochemical immunosensors have been developed to varying degrees along with the development of various disciplines and new technologies. However, owing to the potential instability, volatility, and other shortcomings of biological identification unit (antibody), the generation of suitable electrochemical immunosensors in terms of stability and reproducibility requires further improvement. The rapid development of biology, informatics, materials science and microelectronics will likely provide a better platform for the development of electrochemical immunosensing technology.

Electrochemical Immunosensors Based on Various Nanomaterials
In recent years, with the rapid development of nanotechnology, a variety of new nano-scale materials with excellent performance have been developed, providing a wider platform for the construction of high-performance biosensors [60]. The application of nanomaterials in electrochemical immunosensors mainly includes the following aspects [61,62]: (1) as a sensor substrate for the immobilization of biomolecules to increase the loading amount and further enhance the reactivity; and (2) as an antibody/antigen marker without impairing the activity of the biomolecule and the corresponding components. The concentration of the target analyte can be determined based on the electrochemical detection of nanomaterials and the use of nanostructured amplification markers can greatly increase the signal to produce ultra-sensitive electrochemical immunosensors [63]. To date, various nanomaterials including metal-based (Au, Ag, Pt, or Cu nanoparticles or nanoclusters), carbon-based (CNTs, mesoporous carbon, or graphene), semiconductor (SiO 2 nanoparticles or films, quantum dots (CdTe, PbS or carbon dot), and composite nanomaterials have been applied in the construction of electrochemical immunosensors on different types of signals and have been recognized for their properties of high sensitivity and stability as well as low-cost, thus providing broad application prospects [64][65][66].

Au and Ag Nanomaterials
Au metal is a good conductor of electricity and Au materials exhibit strong adsorption and good biocompatibility, which can be applied for the immobilization of biomolecules and to supply a stable environment to effectively maintain enzyme and other biological protein activity. The Au nanomaterials are mainly divided into AuNPs [67,68], Au nanorods [69,70] and Au nanowires [71], among which AuNPs are of the most interesting, owing to their advantages of simple and rapid preparation, high stability, and uniform particle size [72]. Au nanomaterials are usually immobilized on the surface of the electrode by a bifunctional ligation reagent such as p-thiophenol [73], 1,6-hexanedithiol [74] or polythionine [75] for physical or chemical adsorption of the capture probes. Antibody recognition molecules can also be linked to AuNPs firstly and subsequently fixed to the surface of the electrodetransducer [76,77].
Das et al. [78] prepared a low-noise immunosensor in an indirect determination mode using an AuNPs labeled secondary antibody to catalyze the reduction of p-nitrophenol to electroactive p-aminophenol, and the produced electrical activity aids in detecting the antigen concentration. Simultaneously, the sodium borohydride (NaBH 4 ) was added into the reaction solution to reduce the reaction product. In a similar approach, Tang et al. [79] used AuNPs to functionalize multi-walled CNTs and employed this new AuNPs-multi-walled CNTs material for antibodies labeling, which were further immobilized on the electrode surface to fabricate an electrochemical immunosensor by sandwich immunoassay. This innovative research also employed the substrate cycling reaction of nitrophenol, NaBH 4 and thionine catalyzed by AuNPs, and thus amplifying the response current and greatly improving the detection sensitivity. A diagram illustrating the construction of immunosensors and signal amplification principles is shown in Figure 5. sandwich immunoassay. This innovative research also employed the substrate cycling reaction of nitrophenol, NaBH4 and thionine catalyzed by AuNPs, and thus amplifying the response current and greatly improving the detection sensitivity. A diagram illustrating the construction of immunosensors and signal amplification principles is shown in Figure 5. Furthermore, Sun et al. [80] employed p-dimethylmercaptobenzene as a cross-linking agent to obtain a nanoporous gold (NPG) material with high conductivity and large surface area via a layer by layer self-assembly technique involving the alternate assembly of AuNPs and Ag nanoparticles (AgNPs) on the glassy carbon electrode surface, followed by the use of nitric acid to dissolve the AgNPs ( Figure 6). The NPG material could absorb a large number of electroactive thionines through the Au-S bond and the electrostatic interaction, further enhancing the sensitivity of the immunosensor with a detection limit achieving to 3 pg·mL −1 . Chen et al. [81] has reported a type of ordered 3D-Au nanoclusters generated by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane for the fabrication of an amperometric immunosensor. The electrodeposited Au nanoclusters directly formed electrical contacts and immobilization interfaces with protein molecules without post modification and positioning. Bovine serum albumin-picloram was immobilized by self-assembly on the 3D Au nanoclusters and then competitive immunoreaction with the picloram antibody and target picloram was executed and HRPlabeled secondary antibody was applied for enzyme-amplified amperometric measurement. The proposed amperometric immunosensor has exhibited good precision, sensitivity, selectivity, and storage stability.  Furthermore, Sun et al. [80] employed p-dimethylmercaptobenzene as a cross-linking agent to obtain a nanoporous gold (NPG) material with high conductivity and large surface area via a layer by layer self-assembly technique involving the alternate assembly of AuNPs and Ag nanoparticles (AgNPs) on the glassy carbon electrode surface, followed by the use of nitric acid to dissolve the AgNPs ( Figure 6). The NPG material could absorb a large number of electroactive thionines through the Au-S bond and the electrostatic interaction, further enhancing the sensitivity of the immunosensor with a detection limit achieving to 3 pg·mL −1 . Chen et al. [81] has reported a type of ordered 3D-Au nanoclusters generated by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane for the fabrication of an amperometric immunosensor. The electrodeposited Au nanoclusters directly formed electrical contacts and immobilization interfaces with protein molecules without post modification and positioning. Bovine serum albumin-picloram was immobilized by self-assembly on the 3D Au nanoclusters and then competitive immunoreaction with the picloram antibody and target picloram was executed and HRP-labeled secondary antibody was applied for enzyme-amplified amperometric measurement. The proposed amperometric immunosensor has exhibited good precision, sensitivity, selectivity, and storage stability. sandwich immunoassay. This innovative research also employed the substrate cycling reaction of nitrophenol, NaBH4 and thionine catalyzed by AuNPs, and thus amplifying the response current and greatly improving the detection sensitivity. A diagram illustrating the construction of immunosensors and signal amplification principles is shown in Figure 5. Furthermore, Sun et al. [80] employed p-dimethylmercaptobenzene as a cross-linking agent to obtain a nanoporous gold (NPG) material with high conductivity and large surface area via a layer by layer self-assembly technique involving the alternate assembly of AuNPs and Ag nanoparticles (AgNPs) on the glassy carbon electrode surface, followed by the use of nitric acid to dissolve the AgNPs ( Figure 6). The NPG material could absorb a large number of electroactive thionines through the Au-S bond and the electrostatic interaction, further enhancing the sensitivity of the immunosensor with a detection limit achieving to 3 pg·mL −1 . Chen et al. [81] has reported a type of ordered 3D-Au nanoclusters generated by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane for the fabrication of an amperometric immunosensor. The electrodeposited Au nanoclusters directly formed electrical contacts and immobilization interfaces with protein molecules without post modification and positioning. Bovine serum albumin-picloram was immobilized by self-assembly on the 3D Au nanoclusters and then competitive immunoreaction with the picloram antibody and target picloram was executed and HRPlabeled secondary antibody was applied for enzyme-amplified amperometric measurement. The proposed amperometric immunosensor has exhibited good precision, sensitivity, selectivity, and storage stability.  Notably, some studies [82][83][84] have reported the further enhancement of AuNPs-labeled molecule biometric signal amplification using other nanomaterials, which provide a secondary amplification of detection signal and improve the detection sensitivity significantly. For example, Wang et al. [85] has reported an AgNPs-labeled immunosensor based on MoS 2 -Au composite film for carcino-embryonic antigen (CEA) detection (Figure 7).
Notably, some studies [82][83][84] have reported the further enhancement of AuNPs-labeled molecule biometric signal amplification using other nanomaterials, which provide a secondary amplification of detection signal and improve the detection sensitivity significantly. For example, Wang et al. [85] has reported an AgNPs-labeled immunosensor based on MoS2-Au composite film for carcino-embryonic antigen (CEA) detection (Figure 7). In this study, a MoS2-Au composite film with good catalytic activity toward H2O2 was firstly applied on a glassy carbon electrode and then the second antibody and glucose oxidase were incoporated modified in AgNPs to obtain an AgNPs-Ab2-GOx composite. The whole detection process was performed using differential pulse voltammetry in H2O2 solution containing 1% glucose with a linear range of 1 pg·mL −1 -50 ng·mL −1 and a detection limit of 0.27 pg·mL −1 . Additionally, Chu et al. [86] reported a AgNPs-enhanced colloidal gold-labeled electrochemical immunosensor. The antigen was first immunoreacted with the primary antibody adsorbed onto the polystyrene micropores and then combined with a gold-labeled secondary antibody. After the immune reaction, the AgNPs was added to catalyze the metal reduction to form silver film on the AuNPs. The silver film was then acidolyzed and subjected to electrochemical immunoassay on a glassy carbon electrode by indirect stripping voltammetry. In comparison, Wang et al. [87] synthesized Au-Ag core-shell nanoparticles by an AgNPs-enhanced effect that were used for the detection of platelet-derived growth factor. In the experiment, it was demonstrated that as the increase of the target analyte concentration, more silver metal was deposited on the electrode surface to form a stronger catalytic signal, thus achieving sensitive detection.

Other Metal Nanomaterials
Nanomaterials of other metals including Cu, Pd, Pt and others have also been incorporated for the fabrication of novel electrochemical immunosensor. Various metal nanomaterials have improved the performance of immunosensors, especially in the aspect of signal amplification to enhance the detection sensitivity [88][89][90].
Zhang et al. [91] have synthesized Cu-doped titanium dioxide nanoparticle (Cu@TiO2) and applied it as labels for the fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (Figure 8). The prepared Cu@TiO2 nanocomposite shows high electrocatalytic activity towards reduction of H2O2 at the presence of Cu ions. The dual functionality of Cu@TiO2 enables the fabrication of immunosensor showing high sensitivity, acceptable stability and good reproducibility for both detection modes (square wave voltammetry: linear range: 0.1 pg·mL −1 -100 In this study, a MoS 2 -Au composite film with good catalytic activity toward H 2 O 2 was firstly applied on a glassy carbon electrode and then the second antibody and glucose oxidase were incoporated modified in AgNPs to obtain an AgNPs-Ab 2 -GO x composite. The whole detection process was performed using differential pulse voltammetry in H 2 O 2 solution containing 1% glucose with a linear range of 1 pg·mL −1 -50 ng·mL −1 and a detection limit of 0.27 pg·mL −1 . Additionally, Chu et al. [86] reported a AgNPs-enhanced colloidal gold-labeled electrochemical immunosensor. The antigen was first immunoreacted with the primary antibody adsorbed onto the polystyrene micropores and then combined with a gold-labeled secondary antibody. After the immune reaction, the AgNPs was added to catalyze the metal reduction to form silver film on the AuNPs. The silver film was then acidolyzed and subjected to electrochemical immunoassay on a glassy carbon electrode by indirect stripping voltammetry. In comparison, Wang et al. [87] synthesized Au-Ag core-shell nanoparticles by an AgNPs-enhanced effect that were used for the detection of platelet-derived growth factor. In the experiment, it was demonstrated that as the increase of the target analyte concentration, more silver metal was deposited on the electrode surface to form a stronger catalytic signal, thus achieving sensitive detection.

Other Metal Nanomaterials
Nanomaterials of other metals including Cu, Pd, Pt and others have also been incorporated for the fabrication of novel electrochemical immunosensor. Various metal nanomaterials have improved the performance of immunosensors, especially in the aspect of signal amplification to enhance the detection sensitivity [88][89][90].
The different morphologies of metal nanoparticles could affect their properties and suitability for use. For example, Pt nanoparticles with different morphologies such as hollow versus solid Pt nanospheres display different electrochemical characteristics when used as labels in immunosensors and in other applications [92,93]. Cui et al. [94] have developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. The M-Pt NPs were employed to label the secondary antibody (Ab2) for signal amplification in sandwich-type immunoreactions. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this proposed immunosensor array showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002 U·mL −1 , 0.001 U·mL −1 and 7.0 pg·mL −1 for CA125, CA153 and CEA, respectively.

Carbon-Based Nanomaterials
In recent years, carbon-based nanomaterials derived from a range of forms, from C60, to CNTs to graphene have become the most active research field in nanoscience, effectively promoting the rapid development of nanotechnology [95][96][97]. Carbon-based nanomaterials usually have good electrical conductivity and biocompatibility, and can improve the active sites of electrochemical reactions [98]. Due to the large specific surface area that increases the amount of immobilized biomolecule, this kind of materials are of interest in the field of electrochemical research.

Carbon Nanotubes (CNTs)
Among one-dimensional nanomaterials CNTs have both a special structure (radius: 2-20 nm, with axial dimensions on the micron scale) and a large specific surface area. The carbon atoms in CNTs form a wide range of delocalized bonds with significant conjugation effects, resulting in substantive conductivity. Based on these excellent features, CNTs have a very wide range of applications in immunosensors [99][100][101][102][103]. Among these, the use of CNTs as electrode modifying substrates or markers to build highly sensitive sandwich-type immunosensors represent an important aspect of their study.
Rusling and his colleagues [104] have found that CNTs have good intermolecular electron transfer properties, when they are linked to HRP to modify the electrode. Based on this result, they designed a sandwich electrochemical immunosensor (Figure 9a). Furthermore, a sandwich immunoassay was developed using multi-walled CNTs that included the following steps: (1) CNTs with specific surface area were used to immobilize the primary antibody on the electrode surface; (2) The different morphologies of metal nanoparticles could affect their properties and suitability for use. For example, Pt nanoparticles with different morphologies such as hollow versus solid Pt nanospheres display different electrochemical characteristics when used as labels in immunosensors and in other applications [92,93]. Cui et al. [94] have developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. The M-Pt NPs were employed to label the secondary antibody (Ab 2 ) for signal amplification in sandwich-type immunoreactions. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this proposed immunosensor array showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002 U·mL −1 , 0.001 U·mL −1 and 7.0 pg·mL −1 for CA125, CA153 and CEA, respectively.

Carbon-Based Nanomaterials
In recent years, carbon-based nanomaterials derived from a range of forms, from C 60 , to CNTs to graphene have become the most active research field in nanoscience, effectively promoting the rapid development of nanotechnology [95][96][97]. Carbon-based nanomaterials usually have good electrical conductivity and biocompatibility, and can improve the active sites of electrochemical reactions [98]. Due to the large specific surface area that increases the amount of immobilized biomolecule, this kind of materials are of interest in the field of electrochemical research.

Carbon Nanotubes (CNTs)
Among one-dimensional nanomaterials CNTs have both a special structure (radius: 2-20 nm, with axial dimensions on the micron scale) and a large specific surface area. The carbon atoms in CNTs form a wide range of delocalized bonds with significant conjugation effects, resulting in substantive conductivity. Based on these excellent features, CNTs have a very wide range of applications in immunosensors [99][100][101][102][103]. Among these, the use of CNTs as electrode modifying substrates or markers to build highly sensitive sandwich-type immunosensors represent an important aspect of their study.
Rusling and his colleagues [104] have found that CNTs have good intermolecular electron transfer properties, when they are linked to HRP to modify the electrode. Based on this result, they designed a sandwich electrochemical immunosensor (Figure 9a). Furthermore, a sandwich immunoassay was developed using multi-walled CNTs that included the following steps: (1) CNTs with specific surface area were used to immobilize the primary antibody on the electrode surface; (2) a sandwich reaction was performed to capture enzyme-labeled secondary antibodies loaded on CNTs on the electrode surface; (3) the addition of H 2 O 2 to the substrate to reflect the concentration of the antigen. Owing to the synergetic effect of HRP and CNTs, the electrochemical response signal of the relative immunosensors is greatly enhanced. The detection limit can reach 4 ng·L −1 for the prostate cancer marker PSA and 40 fg·mL −1 for 10 µL undiluted bovine serum. Jeong et al. [105] employed the HRP and glucose oxidase to label an anti-CEA antibody and immobilize the labeled-antibody on multi-walled CNTs activated by dimethylaminopropyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Use of the multi-walled CNT material significantly increased the immobilized amount of antibody (Figure 9b). This functionalized multiwalled CNT complex was used as a dendritic nanostructure matrix and AuNPs in combination with thionine media to develop a highly sensitive method for the determination of CEA by electrochemical immunoassay. In addition, Liu et al. [106] developed a functional single-walled CNT material for immobilizing antibodies on the surface of a glassy carbon electrode (Figure 9c). The fabricated electrochemical immunosensor could be successfully used for the detection of endosulfan over the range of 0.01-20 ppb with a detection limit of 0.01 ppb in 50 mM phosphate buffer at pH 7.0. Furthermore, Wang et al. [107] employed CNTs as a carrier for covalently bonding a large number of enzyme molecules (approximately 1 μm CNT, representing a load of approximately 9600 enzyme molecules) to prepare an electrochemical immunosensor using CNTs as a marker with a detection limit of 500 fg·mL −1 (160 μL in 25 μL sample). Compared with the ordinary enzyme-labeled electrochemical immunosensor, the signal response of this new electrochemical immunosensor can be improved by 100-fold. Using the displacement mode, trinitrobenzene (TNT) was first applied in Figure 9. The working principle of this typical CNTs-based immunosensor: (a) CNTs-linked HRP for electrode modification. Reproduced with permission from reference [104]. Copyright 2006 American Chemical Society; (b) Activated multi-walled CNTs for immobilizing labeled-antibody. Reproduced with permission from reference [105]. Copyright 2013 American Chemical Society; (c) Single-walled CNT for immobilizing an antibody. Reproduced with permission from reference [106]. Copyright 2012 American Chemical Society.
Jeong et al. [105] employed the HRP and glucose oxidase to label an anti-CEA antibody and immobilize the labeled-antibody on multi-walled CNTs activated by dimethylaminopropyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Use of the multi-walled CNT material significantly increased the immobilized amount of antibody (Figure 9b). This functionalized multi-walled CNT complex was used as a dendritic nanostructure matrix and AuNPs in combination with thionine media to develop a highly sensitive method for the determination of CEA by electrochemical immunoassay. In addition, Liu et al. [106] developed a functional single-walled CNT material for immobilizing antibodies on the surface of a glassy carbon electrode (Figure 9c). The fabricated electrochemical immunosensor could be successfully used for the detection of endosulfan over the range of 0.01-20 ppb with a detection limit of 0.01 ppb in 50 mM phosphate buffer at pH 7.0. Furthermore, Wang et al. [107] employed CNTs as a carrier for covalently bonding a large number of enzyme molecules (approximately 1 µm CNT, representing a load of approximately 9600 enzyme molecules) to prepare an electrochemical immunosensor using CNTs as a marker with a detection limit of 500 fg·mL −1 (160 µL in 25 µL sample). Compared with the ordinary enzyme-labeled electrochemical immunosensor, the signal response of this new electrochemical immunosensor can be improved by 100-fold. Using the displacement mode, trinitrobenzene (TNT) was first applied in the SWCNT conduction channel, and then the anti-TNT antibody was attached [108]. When acting with the target or its derivative, the detection of analyte was achieved by monitoring the change of impedance or conductance owing to the displacement reaction. Notably, this novel immunoassay method using the displacement reaction has a relatively wide detection range (0.5-5000 µg·L −1 TNT).

Graphene and Graphene Oxide
Graphene exhibits unique physical and chemical properties, in particular a monolithic structure, high conductivity, large specific surface area, no toxicity and good electron mobility, and is widely used in the fields of electrochemical sensing and biosensing [109][110][111][112]. The graphene materials carries a high density of defects on the surface and demonstrates particularly impressive positive electrochemical properties [113]. To date, graphene-modified electrodes have been successfully applied for assessing H 2 O 2 [114], NADH, dopamine, ascorbic acid, uric acid and acetaminophen. The application of graphene onto a variety of inorganic and organic electroactive materials is further illustrated by its role as a promising new carbon substrate in the field of electrochemical analysis.
Narayanan et al. [115] strongly modified graphene on the surface of a glassy carbon electrode by a series of covalent reactions, and an antibody was further modified on the graphene using the EDC/NHS cross-linking reagent. Using RaMIgG-ALP/AuNPs as a signal amplification marker, the detection of botulinum neurotoxin E was achieved through sandwich immunization with the detection range and detection limit achieving to 10 pg·mL −1 -10 ng·mL −1 and 5.0 pg·mL −1 , respectively. Figure 10 shows an electrochemical method developed by Sharma et al. [116] applied to reduce the graphene on the surface of a screen printed electrode and develop a sandwich electrochemical immunosensor for the detection of diuron. Furthermore, the graphene can also be applied for the construction of immune probes for detection [117]. For example, Li et al. [118] has prepared an Ru-AuNPs/graphene probe for CEA detection. The Ru-AuNPs/graphene probe was synthesized layer by layer using AuNPs, Ru(bpy) 3 2+ and poly(dimethyldiallylammonium chloride)-pretreated graphene and further reacted with an antibody immobilized on a nanoporous Au membrane. Yang et al. [119] also fabricated an immune-labeled probe using graphene as a carrier of thionine and HRP to prepare a sandwich electrochemical immunosensor for the detection of tumor markers. the SWCNT conduction channel, and then the anti-TNT antibody was attached [108]. When acting with the target or its derivative, the detection of analyte was achieved by monitoring the change of impedance or conductance owing to the displacement reaction. Notably, this novel immunoassay method using the displacement reaction has a relatively wide detection range (0.5-5000 μg·L −1 TNT).

Graphene and Graphene Oxide
Graphene exhibits unique physical and chemical properties, in particular a monolithic structure, high conductivity, large specific surface area, no toxicity and good electron mobility, and is widely used in the fields of electrochemical sensing and biosensing [109][110][111][112]. The graphene materials carries a high density of defects on the surface and demonstrates particularly impressive positive electrochemical properties [113]. To date, graphene-modified electrodes have been successfully applied for assessing H2O2 [114], NADH, dopamine, ascorbic acid, uric acid and acetaminophen. The application of graphene onto a variety of inorganic and organic electroactive materials is further illustrated by its role as a promising new carbon substrate in the field of electrochemical analysis.
Narayanan et al. [115] strongly modified graphene on the surface of a glassy carbon electrode by a series of covalent reactions, and an antibody was further modified on the graphene using the EDC/NHS cross-linking reagent. Using RaMIgG-ALP/AuNPs as a signal amplification marker, the detection of botulinum neurotoxin E was achieved through sandwich immunization with the detection range and detection limit achieving to 10 pg·mL −1 -10 ng·mL −1 and 5.0 pg·mL −1 , respectively. Figure 10 shows an electrochemical method developed by Sharma et al. [116] applied to reduce the graphene on the surface of a screen printed electrode and develop a sandwich electrochemical immunosensor for the detection of diuron. Furthermore, the graphene can also be applied for the construction of immune probes for detection [117]. For example, Li et al. [118] has prepared an Ru-AuNPs/graphene probe for CEA detection. The Ru-AuNPs/graphene probe was synthesized layer by layer using AuNPs, Ru(bpy)3 2+ and poly(dimethyldiallylammonium chloride)-pretreated graphene and further reacted with an antibody immobilized on a nanoporous Au membrane. Yang et al. [119] also fabricated an immune-labeled probe using graphene as a carrier of thionine and HRP to prepare a sandwich electrochemical immunosensor for the detection of tumor markers. Figure 10. Illustration of in-situ electrochemical synthesis of functionalized graphene-graphene oxide nanocomposite on screen printed electrodes and subsequent immunoassay development. Reproduced with permission from reference [116]. Copyright 2013 Elsevier.
With the development of graphene materials, many researchers have introduced or removed some functional groups on their surface to obtain graphene derivatives such as graphene oxide and reduced graphene [120]. Specifically, oxygen-containing functional groups are introduced into the graphene to obtain graphene oxide; subsequently, incomplete removal of these groups by chemical or heat treatment is used to obtain reduced graphene. The resulting graphene derivatives with different properties also have good applications in the field of immunosensors. In particular, Liu et al. [121] designed an immunosensor for the simultaneous detection of CEA and AFP, in which With the development of graphene materials, many researchers have introduced or removed some functional groups on their surface to obtain graphene derivatives such as graphene oxide and reduced graphene [120]. Specifically, oxygen-containing functional groups are introduced into the graphene to obtain graphene oxide; subsequently, incomplete removal of these groups by chemical or heat treatment is used to obtain reduced graphene. The resulting graphene derivatives with different properties also have good applications in the field of immunosensors. In particular, Liu et al. [121] designed an immunosensor for the simultaneous detection of CEA and AFP, in which graphene oxide was applied as the platform for immunoreaction. Reduced graphene [122] has also been applied for the fabrication of an immunosensing platform for CEA and AFP. The composites of multi-walled CNTs [123] and graphene provide a 3D electrode interface, which has rich reaction sites for electrochemical reactions, enhancing the electron transfer ability between the active sites of the enzyme and the electrode, and thus improving the detection sensitivity. The AuNPs-dotted CNTs-graphene composite and functionalized mesoporous materials (MCM-41) developed by Lu et al. [124] also significantly enhanced the detection sensitivity of electrochemical immunosensors (Figure 11). graphene oxide was applied as the platform for immunoreaction. Reduced graphene [122] has also been applied for the fabrication of an immunosensing platform for CEA and AFP. The composites of multi-walled CNTs [123] and graphene provide a 3D electrode interface, which has rich reaction sites for electrochemical reactions, enhancing the electron transfer ability between the active sites of the enzyme and the electrode, and thus improving the detection sensitivity. The AuNPs-dotted CNTsgraphene composite and functionalized mesoporous materials (MCM-41) developed by Lu et al. [124] also significantly enhanced the detection sensitivity of electrochemical immunosensors ( Figure 11).

Other Carbon Materials
Carbon nanofibers have a greater functional surface area and more surface active groups, and thus are considered to be a more promising material [125,126]. Carbon nanospheres have relatively richer functional groups with better biocompatibility, dispersibility, and relative activity [127].
Wu et al. [128] first used soluble carbon fiber to fabricate an immunosensor with rapid free isolation. Cui et al. [129] prepared carbon nanospheres using a microwaved hot solution method and further modified the surface of carbon nanospheres with the nano-gold to obtain composite nanometer microspheres with high surface energy and good biocompatibility. The labeled secondary antibody was labeled on the surface of gold-carbon composite nanospheres to obtain highly sensitive electrochemical immunosensors that achieved to a detection limit of 5.6 pg·mL −1 . In addition, Du et al. [130] used gravimetric and functionalized carbon nanospheres to double the amplification of the electrochemical signal, resulting in a detection signal of the developed immunosensor 7-fold greater than that of normal sensors.

Semiconductor Nanomaterials
Semiconductor nanomaterials primarily including silica nanoparticles and quantum dots, exhibit excellent properties such as large specific surface area, high surface reaction activity, and strong adsorption capacity, providing a new route for biomedical research.

SiO2 Nanomaterials
The surface coordination of nano-SiO2 particles present with a large number of unsaturated residues and different bonds and hydroxyl groups, allowing this materials to be readily surfacefunctionalized. As they also exhibit good biocompatibility toward biologically active molecules, nano-SiO2 particles represent excellent carriers for the immobilization of proteins and enzymes in electrochemical biosensors [131]. For example, Hong et al. [132] prepared ferrocene-coated SiO2 Figure 11. Fabrication process of AuNPs/Thionine/MCM-41 nanomaterials and measurement protocol of the electrochemical immunosensor. Reproduced with permission from reference [124]. Copyright 2012 Elsevier.

Other Carbon Materials
Carbon nanofibers have a greater functional surface area and more surface active groups, and thus are considered to be a more promising material [125,126]. Carbon nanospheres have relatively richer functional groups with better biocompatibility, dispersibility, and relative activity [127].
Wu et al. [128] first used soluble carbon fiber to fabricate an immunosensor with rapid free isolation. Cui et al. [129] prepared carbon nanospheres using a microwaved hot solution method and further modified the surface of carbon nanospheres with the nano-gold to obtain composite nanometer microspheres with high surface energy and good biocompatibility. The labeled secondary antibody was labeled on the surface of gold-carbon composite nanospheres to obtain highly sensitive electrochemical immunosensors that achieved to a detection limit of 5.6 pg·mL −1 . In addition, Du et al. [130] used gravimetric and functionalized carbon nanospheres to double the amplification of the electrochemical signal, resulting in a detection signal of the developed immunosensor 7-fold greater than that of normal sensors.

Semiconductor Nanomaterials
Semiconductor nanomaterials primarily including silica nanoparticles and quantum dots, exhibit excellent properties such as large specific surface area, high surface reaction activity, and strong adsorption capacity, providing a new route for biomedical research.

SiO 2 Nanomaterials
The surface coordination of nano-SiO 2 particles present with a large number of unsaturated residues and different bonds and hydroxyl groups, allowing this materials to be readily surface-functionalized. As they also exhibit good biocompatibility toward biologically active molecules, nano-SiO 2 particles represent excellent carriers for the immobilization of proteins and enzymes in electrochemical biosensors [131]. For example, Hong et al. [132] prepared ferrocene-coated SiO 2 nanoparticles using oil-in-water technology to form stable, homogeneous, biocompatible, and highly surface active interfaces to effectively immobilize the antibodies in a no-reagent electrochemical immunosensor. In particular, this sensor had simple preparation, low cost and high sensitivity characteristics. Figure 12 shows a schematic diagram of a novel electrochemical immunosensor reported by Wu et al. [133] based on NPG and hollow mesoporous silica microspheres (HSMs). Owing to the large specific surface area of amino-functionalized HSMs, the loading amount of antibodies as well as mediators and enzymes thereon was substantially increased, which in turn increased the sensitivity of the immunosensor (limit of detection: 6.00 pg·mL −1 ). This new method may be quite promising with potential broad applications for clinical immunoassays. Additionally, Yang et al. [134] first used a porous silicon ball to adsorb a large amount of thionine, in addition to a large amount of HRP and signal antibodies by covalent loading. Through the catalytic signal amplification from the HRP enzyme, highly sensitive detection of the cancer markers was achieved. Furthermore, the researchers in this group applied a similar method for modifying an ionic liquid onto a porous silicon sphere surface for biomolecules immobilization. Combined with the sandwich immunoassay, an amperometric immunosensor was thereby successfully fabricated for highly sensitive detection of the cancer markers [135]. nanoparticles using oil-in-water technology to form stable, homogeneous, biocompatible, and highly surface active interfaces to effectively immobilize the antibodies in a no-reagent electrochemical immunosensor. In particular, this sensor had simple preparation, low cost and high sensitivity characteristics. Figure 12 shows a schematic diagram of a novel electrochemical immunosensor reported by Wu et al. [133] based on NPG and hollow mesoporous silica microspheres (HSMs). Owing to the large specific surface area of amino-functionalized HSMs, the loading amount of antibodies as well as mediators and enzymes thereon was substantially increased, which in turn increased the sensitivity of the immunosensor (limit of detection: 6.00 pg·mL −1 ). This new method may be quite promising with potential broad applications for clinical immunoassays. Additionally, Yang et al. [134] first used a porous silicon ball to adsorb a large amount of thionine, in addition to a large amount of HRP and signal antibodies by covalent loading. Through the catalytic signal amplification from the HRP enzyme, highly sensitive detection of the cancer markers was achieved. Furthermore, the researchers in this group applied a similar method for modifying an ionic liquid onto a porous silicon sphere surface for biomolecules immobilization. Combined with the sandwich immunoassay, an amperometric immunosensor was thereby successfully fabricated for highly sensitive detection of the cancer markers [135]. Tang et al. [136] has added the complex of thionine and HRP enzyme into nano-silica solution, further combining this with an HRP-labeled secondary antibody to construct an electrochemical immunosensor. In this study, the electron mediator and the enzyme label were both modified on the sensor recognition element, simplifying the electrochemical measurement process and resulting in a high detection sensitivity toward the target cancer marker CA125 (limit of detection: 0.1 U·mL −1 ). Wu et al. [137] also prepared a uniform dispersion of silica nanospheres and silane-functionalized surface for labeling signal antibodies and loading high levels of HRP enzyme. This allowed a highly sensitive immunoassay for AFP to be developed in combination with sandwich mode and enzyme-catalyzed cyclic amplification. When the antibody and quantum dots were simultaneously applied onto the surface of the silica nanospheres, this composite signal tracer could greatly improve the electrochemical signal combined with metal ion electrochemical dissolution analysis, achieving highly sensitive detection of the target analytes [138,139].

Quantum Dots
A variety of quantum dots not only have the characteristics of nanomaterials, but also can give good, sharp and sensitive dissolution peaks through electrochemical dissolution voltammetry [140][141][142]. Thus, quantum dot materials have good prospects in the related fields of nanomaterials and electrochemical analysis [143][144][145].
Liu et al. [146] have used different semiconductor nanomaterials (ZnS, CdS, PbS and CuS) for labeling various proteins (α2-microglobulin, IgG, bovine serum albumin, C-reactive protein). Tang et al. [136] has added the complex of thionine and HRP enzyme into nano-silica solution, further combining this with an HRP-labeled secondary antibody to construct an electrochemical immunosensor. In this study, the electron mediator and the enzyme label were both modified on the sensor recognition element, simplifying the electrochemical measurement process and resulting in a high detection sensitivity toward the target cancer marker CA125 (limit of detection: 0.1 U·mL −1 ). Wu et al. [137] also prepared a uniform dispersion of silica nanospheres and silane-functionalized surface for labeling signal antibodies and loading high levels of HRP enzyme. This allowed a highly sensitive immunoassay for AFP to be developed in combination with sandwich mode and enzyme-catalyzed cyclic amplification. When the antibody and quantum dots were simultaneously applied onto the surface of the silica nanospheres, this composite signal tracer could greatly improve the electrochemical signal combined with metal ion electrochemical dissolution analysis, achieving highly sensitive detection of the target analytes [138,139].

Quantum Dots
A variety of quantum dots not only have the characteristics of nanomaterials, but also can give good, sharp and sensitive dissolution peaks through electrochemical dissolution voltammetry [140][141][142]. Thus, quantum dot materials have good prospects in the related fields of nanomaterials and electrochemical analysis [143][144][145].
Liu et al. [146] have used different semiconductor nanomaterials (ZnS, CdS, PbS and CuS) for labeling various proteins (α2-microglobulin, IgG, bovine serum albumin, C-reactive protein). According to the peak position of the marker and the tested peak current from the stripping voltammetry, different antigens were identified and determined. Figure 13 shows the analytical procedure of a sandwich-type immunosensor fabricated by Cui et al. [147] on an ITO chip covered with a well-ordered AuNPs monolayer applying CdTe quantum dots as electrochemical and fluorescent labels. The detection sensitivity could be increased to 0.005 ng·mL −1 with the linear range of 0.005-100 ng·mL −1 by stripping voltammetric analysis. Furthermore, Huang et al. [148] applied a composite film of Au@carbon dots-chitosan in the modification of a glassy carbon electrode for the sensitive and reliable determination of dopamine. The carbon dots contained carboxyl groups with negative charge, which not only provided it with good stability but also enabled the interaction with amine functional groups in dopamine through electrostatic interaction, resulting in multiple-recognition of dopamine with high specificity. According to the peak position of the marker and the tested peak current from the stripping voltammetry, different antigens were identified and determined. Figure 13 shows the analytical procedure of a sandwich-type immunosensor fabricated by Cui et al. [147] on an ITO chip covered with a well-ordered AuNPs monolayer applying CdTe quantum dots as electrochemical and fluorescent labels. The detection sensitivity could be increased to 0.005 ng·mL −1 with the linear range of 0.005-100 ng·mL −1 by stripping voltammetric analysis. Furthermore, Huang et al. [148] applied a composite film of Au@carbon dots-chitosan in the modification of a glassy carbon electrode for the sensitive and reliable determination of dopamine. The carbon dots contained carboxyl groups with negative charge, which not only provided it with good stability but also enabled the interaction with amine functional groups in dopamine through electrostatic interaction, resulting in multiple-recognition of dopamine with high specificity.

Other Nanomaterials
Magnetic microspheres represent a new type of magnetic material or nanomaterial developed in recent years. Compared with ordinary magnetic microspheres, magnetic composite microspheres exhibit good magnetic response and nano-synergistic effects in addition to the many characteristics of ordinary microspheres (such as a large surface area), and can also provide more functional groups such as -OH, -NH2, -COOH, and -CHO on their surfaces. Some studies have reported the use of magnetic composite nanomaterials in electrochemical immunosensors [149].
Zhou et al. [150] and Tang et al. [151] have reported using magnetic gold nanospheres doped with thionine-labeled HRP and signal antibodies and Prussian blue-labeled HRP-glucose oxidase and signal antibodies to prepare two immunosensors for sensitive detection of CEA. Ding et al. [152] employed streptavid in functionalized magnetic beads to fix a large number of antibodies (Ab1) on the sensor surface for the combination with AuNPs-labeled antibodies (Ab2) and quantum dots. They thus formed a homogeneous sandwich immunoassay to achieve AFP detection through the analysis of stripping voltammetry of quantum dots. A similar immunosensor [153] was also developed for prostate antigen detection by immobilizing Ab1 using carboxylated magnetic nanospheres.
Furthermore, Feng et al. [154] applied metal NPs-functionalized phosphopeptide nanospheres to label Ab2 as a signal nanoprobe ( Figure 14). The antibody (Ab1) was immobilized by the oxidized graphene activated by EDC/NHS on the surface of the electrode. The developed sandwich electrochemical immunoassay method can simultaneously determine three proteins: AFP, cardiac troponin I and human myocardial fatty acid binding protein.

Other Nanomaterials
Magnetic microspheres represent a new type of magnetic material or nanomaterial developed in recent years. Compared with ordinary magnetic microspheres, magnetic composite microspheres exhibit good magnetic response and nano-synergistic effects in addition to the many characteristics of ordinary microspheres (such as a large surface area), and can also provide more functional groups such as -OH, -NH 2 , -COOH, and -CHO on their surfaces. Some studies have reported the use of magnetic composite nanomaterials in electrochemical immunosensors [149].
Zhou et al. [150] and Tang et al. [151] have reported using magnetic gold nanospheres doped with thionine-labeled HRP and signal antibodies and Prussian blue-labeled HRP-glucose oxidase and signal antibodies to prepare two immunosensors for sensitive detection of CEA. Ding et al. [152] employed streptavid in functionalized magnetic beads to fix a large number of antibodies (Ab1) on the sensor surface for the combination with AuNPs-labeled antibodies (Ab2) and quantum dots. They thus formed a homogeneous sandwich immunoassay to achieve AFP detection through the analysis of stripping voltammetry of quantum dots. A similar immunosensor [153] was also developed for prostate antigen detection by immobilizing Ab1 using carboxylated magnetic nanospheres.
Furthermore, Feng et al. [154] applied metal NPs-functionalized phosphopeptide nanospheres to label Ab2 as a signal nanoprobe ( Figure 14). The antibody (Ab1) was immobilized by the oxidized graphene activated by EDC/NHS on the surface of the electrode. The developed sandwich electrochemical immunoassay method can simultaneously determine three proteins: AFP, cardiac troponin I and human myocardial fatty acid binding protein.

Conclusions
In recent years, various kinds of nanomaterials with different properties have been synthesized, and their applications in electrochemical immunosensor have become increasingly popular, thus leading to a current research hotspot. According to the current status of related research, more indepth studies are anticipated regarding the following aspects of nanomaterials application in electrochemical immunosensor: (1) As biomarkers. More electrochemical markers from novel nanofunctional materials will continue to emerge, which will further improve the sensitivity of immunoassays. It is believed that with the development of science and technology and the expansion of related research, a greater number of new nanomaterials with high performance will be applied to electrochemical immunosensors, thus providing broad prospects for their development.

Conclusions
In recent years, various kinds of nanomaterials with different properties have been synthesized, and their applications in electrochemical immunosensor have become increasingly popular, thus leading to a current research hotspot. According to the current status of related research, more in-depth studies are anticipated regarding the following aspects of nanomaterials application in electrochemical immunosensor: (1) As biomarkers. More electrochemical markers from novel nanofunctional materials will continue to emerge, which will further improve the sensitivity of immunoassays. It is believed that with the development of science and technology and the expansion of related research, a greater number of new nanomaterials with high performance will be applied to electrochemical immunosensors, thus providing broad prospects for their development.


Recombination via transition metals in solar silicon: The significance of hydrogen–metal reactions and lattice sites of metal atoms

The move toward lower cost sources of solar silicon has intensified the efforts to investigate the possibilities of passivating or reducing the recombination activity caused by deep states associated with transition metals. This is particularly important for the case of the slow diffusing metals early in the periodic sequence which are not removed by conventional gettering. In this paper, we examine reactions between hydrogen and transition metals and discuss the possibility of such reactions during cell processing. We analyse the case of hydrogenation of iron in p‐type Si and show that FeH can form under non‐equilibrium conditions. We consider the electrical activity of the slow diffusing metals Ti, V, and Mo, how this is affected in the presence of hydrogen, and the stability of TM‐H complexes formed. Finally, we discuss recent experiments which indicate that re‐siting of some transition metals from the interstitial to substitutional site is possible in the presence of excess vacancies, leading to a reduction in recombination activity.

1 Introduction In silicon technologies much attention has been devoted to contamination by transition metals (TM), particularly those in the 3d series. These metals are responsible for yield reducing problems associated with, for example, generation currents in MOS devices and dielectric breakdown [1]. In the case of device fabrication using electronic grade silicon, the concern is largely associated with metals introduced during device processing, electronic grade silicon in its as-grown state contains negligible concentrations of transition metals. This is because it derives from polycrystalline feedstock which has been chemically purified very effectively, for example by the Siemens process, and then is converted to single crystal from the melt usually by the Czochralski (Cz) method. The effective segregation coefficient of transition metals between the solid and the liquid phase of the silicon during Cz growth is typically <10 À5 resulting in most of the TMs remaining in the melt rather than being transferred to the grown crystal. The combination of the Siemens process and Cz growth usually results in extremely low and technologically unimportant concentrations of TMs, typically these are 10 10 cm À3 .
In the case of Solar Silicon, the cost of the silicon is much more important than in the case of electronic grade material applications in microelectronics and so cheaper methods of ingot growth are often used. Many variants on the theme are in production or in development but the end result is that the chemical purification of the feedstock is simplified, for example upgraded metallurgical silicon (UMG) is used, or the inherent purification of the Czochralski method is eliminated using casting. This results in a higher concentration of transition metals in the slices used for solar cell fabrication than in electronic grade Si.
The significance of specific TMs in solar cells was studied in the 1970's in what is now known as "The Westinghouse Experiment." In this work, various concentrations of TMs were added to the melt in a conventional Cz growth procedure and the effect on the solar cell efficiency was measured. The study was an extremely comprehensive one, involving the growth of over 200 TM doped Cz ingots in which the chemical concentrations were measured using neutron activation analysis (NAA), the electrical activity using deep level transient spectroscopy (DLTS), and the detailed effect on the solar cell performance monitored [2].
Families of graphs were published showing the degradation of efficiency as a function of the TM concentration in the p and n-type Si slices used to fabricate the cells, and this was widely referenced showing that certain metals were the most "dangerous." When the results are analysed, it becomes apparent that the order of significance is, in general, the order of the TMs diffusivities (fast diffusers are the least significant in degrading the cell efficiency) and is not solely related to the recombination activity of specific transition metals as was originally proposed [3]. The cell fabrication process used in the Westinghouse experiment used single crystal material and included a very effective implementation of gettering. A similar sequence of experiments on multi-crystalline silicon was undertaken more recently and again, slow diffusers were found to be the most pernicious [4,5]. The essential conclusion is that contamination with the slow diffusing TMs is particularly detrimental. These correspond to the lower atomic number elements in the 3d and 4d series sometimes referred to as the early 3d and 4d elements. If high efficiencies are to be obtained with less pure silicon which contains these slow diffusing TMs it is important to find ways in which their recombination activity is reduced or eliminated in both single crystal and multi-crystalline materials.
It is well known that reactions between hydrogen and some TMs in silicon can result in passivation or reduced recombination activity. In general, the bonding between hydrogen and the TM is weak ($2 eV) and so the hydrogen is dissociated at temperatures well below those experienced in solar cell processing. In addition, in p-type material hydrogen-TM reactions are usually inhibited by Coulombic repulsion. Despite this, recent work has indicated that substantial improvements in lifetime of both Cz and cast UMG p-type solar silicon can be achieved through hydrogen treatments in which light is used. This is interpreted as being due to a change in the charge state of the hydrogen [6]. In this paper, we consider these issues and present some of our recent work on the hydrogenation of TMs and also, evidence for displacement of some TMs from the interstitial to substitutional site. In conjunction with earlier work on hydrogenation we try to draw generalisations in relation to the mechanism of hydrogen passivation and its stability. Our principal tools in these studies have been DLTS and related analytical techniques which we describe elsewhere [7].
2 Formation and thermal stability of hydrogen-TM complexes in silicon 2.1 TM-H reactions in p-type Si Hydrogen atoms are very mobile in silicon at room temperature and atomic hydrogen is likely to combine with impurities and intrinsic defects [8]. The initial hydrogen reaction is highly dependent on the charge state relative to the defect or impurity. Predominantly, the reaction is driven by long range Coulombic interactions. Atomic hydrogen is an amphoteric impurity with negative-U ordered donor and acceptor levels at E c À 0.18 eV and $E c À 0.65 eV. The neutral state is metastable. Isolated hydrogen occurs in the positive charge state in p-type Si, while it is negatively charged in n-type. As the H (þ/À) occupancy level lies in the upper half of the band gap it would be expected that in intrinsic Si (mid-gap Fermi level) the H þ charge state will dominate. Experimentally, there is evidence that H À is the stable form when the Fermi level is >0.3 eV above the mid gap energy [9].
The fact that hydrogen is positively charged in p-type Si explains why there are many reports of TM-H complexes in n-type Si, whereas there are none in equilibrium in p-type Si. The interstitial TMs tend to be positively charged in p-type and hence there will be Coulombic repulsion between the metal and the hydrogen. Under these conditions the metalhydrogen complex will not form. However, if the Fermi level can be manipulated or an adequate flux of minority carriers generated, non-equilibrium charge states can be attained and some reactions with H may now become possible.
We have explored this concept by forcing a hydrogen reaction with iron in p-type Si. Previously a Fe i H complex has been characterised in n-type Si as a hole trap with an energy level at 0.31 eV above the valence band [10]. Ab initio calculations have predicted that the Fe i H complex should be stable at room temperature and have a deep donor level at about E v þ 0.40 eV and a deep acceptor level at about E c -0.3 eV [11].
We took Fe contaminated 12 Ω Á cm p-type Cz-Si samples and introduced hydrogen either from a hydrogen-rich silicon nitride layer or by wet etching [12]. The DLTS spectrum for a Fe contaminated and hydrogenated sample is shown in Fig. 1 as line 1, as measured on a Schottky diode evaporated onto an unheated sample. A peak with its maximum at about 55 K, clearly identifiable as related to the Fe-B pair, is evident. If the sample is annealed at 110 8C with zero bias a small peak with its maximum at about 245 K due to Fe i appears (line 2). If, however, the same anneal is undertaken with the Schottky diode under 5 V reverse bias in the DLTS spectrum (line 3) the Fe i related signal becomes much more prominent and a trap with characteristics identical to that ascribed to Fe i H in ntype Si is seen. This is because the 5 V reverse bias establishes a near intrinsic depletion region in which the defect reactions occur. This region is probed in subsequent DLTS scans. Many things are happening during this anneal including the drift of the hydrogen in the electric field toward the bulk of the semiconductor. This can be measured by observing the passivation of the boron acceptors deeper in the bulk using traditional CV measurements. This would indicate that the hydrogen remains positively charged in the depletion region as expected from the fact that the hydrogen converts to H À only when the Fermi level rises to 0.3 eV above the mid gap energy [9]. Fe i with its donor level at about 0.4 eV above the valence band becomes neutral in the depletion region and so, that switches off the attractive Coulomb field of B À for Fe i þ occurring in the bulk. Now, the neutral Fe atoms escaping from the boron at 110 8C are not recaptured by boron (this explains the large increase in the Fe i related DLTS signal in the spectrum 3 in Fig. 1) and are free to interact with H þ .
If this interpretation is correct, then it is evident that whether or not the pairing reaction occurs depends on the charge state of the TM in the depletion region in p-type Si which depends on its 0/þ energy level position and so is species specific.
Titanium and vanadium interstitial atoms have their first donor levels in the upper half of the Si gap at about E c -0.27 eV and E c -0.45 eV, respectively [3]. As the Ti i and V i atoms are always positively charged in p-type Si, the interactions of these atoms with positively charged H atoms are very unlikely in p-type material. Indeed, we have not observed any changes in the DLTS spectra of p-Si crystals, which were contaminated with Ti and V impurities, after hydrogenation [13,14].
The case of Mo contaminated p-type Si crystals is more complicated. Molybdenum is arguably the most potentially harmful metallic impurity considered in this work, seen to significantly reduce carrier lifetime and correspondingly cell efficiency at lower concentrations than Ti and V [2]. It is also practically impossible to getter [15] and has not been observed to form large precipitates, although recent work [16] has shown nanoscale precipitates of Mo can form and act as very efficient recombination centers.
The majority of electrically active Mo in silicon is found at an interstitial site and is known to introduce one deep donor level in the silicon band gap close to E v þ 0.30 eV [2,3]. Some evidence has been presented of Mo-related levels in the upper half of the gap [17,18]. However, the most recent study of these levels [18] suggests that these are not likely to be due to the Mo interstitial. In previous DLTS studies of Mo in p-type material, as well as in our work to date, no evidence of Mo-H complexes has been observed. However, the unidentified DLTS signals related to Mo in the upper half of the gap, which were observed in Refs. [17,18], could be related to Mo-H complexes.
It is to be expected that a shift in Fermi level and hence modification of the charge state could be achieved through illumination with above band gap light so generating minority carriers as implemented in Ref. [6]. However, the situation is much more complex than the Fermi level shift due to changes in bias in our experiments above. This is primarily due to the presence of both minority and majority carriers and their direct reactions with the defects to produce non-equilibrium charge states.

TM-H reactions in n-type Si
In n-type material hydrogen atoms are negatively charged and can travel along the lattice through sites with low electron density (tetrahedral and hexagonal sites). In this situation, TM interstitials are likely to be either neutral or positively charged, increasing the probability of TM-H reactions. A few examples of such interactions are presented below.

Titanium
Interstitial titanium in silicon, located at the tetrahedral interstitial lattice site, presents three levels in the silicon band gap at E c -0.08 eV, E c -0.27 eV, and E v þ 0.26 eV [3]. Despite early results suggesting that hydrogen passivation would only be effective for fast diffusing transition metals, recent studies have shown the formation of Ti-H n complexes in n-type Si [13,19,20].
In our recent work [13], samples of n-type Si implanted with Ti and annealed were treated with a remote hydrogen plasma at room temperature. In these conditions, titanium was shown to form at least three Ti-H n complexes with electrically active levels in the silicon band gap, with some formation of electrically inactive complexes. When annealed at relatively low temperatures (100-150 8C), the concentration of these electrically active Ti and Ti-H n complexes was reduced until almost complete passivation of Ti was observed. DLTS spectra of Ti implanted n-type Cz Si are shown in Fig 2 before and after hydrogenation and subsequent annealing. In the DLTS spectrum of asimplanted samples (spectrum 1 in Fig. 2), two strong peaks with their maxima at about 40 and 150 K corresponding to interstitial Ti are seen. After a treatment with a hydrogen plasma (spectrum 2), new signals corresponding to various Ti-H n complexes are observed modifying the shape of the 150 K peak and are components of the peak at $ 270 K. Crucially, after annealing the hydrogenated samples at 100-150 8C (spectra 3 and 4) all of the Ti and Ti-H n related signals in DLTS are significantly reduced, indicating the formation of an electrically inactive Ti-H complex with no levels in the Si band gap. These complexes are unstable at higher temperatures and nearly a complete recovery of all electrically active Ti was seen after annealing at 250 8C.

Vanadium
Interstitial vanadium, like titanium, is situated at the tetrahedral interstitial lattice site and can cause severe reductions in cell efficiencies at concentrations as low as 10 12 cm À3 . It presents three deep level states in the silicon band gap at E c -0.20 eV, E c -0.45 eV, and E v þ 0.34 eV [3]. In n-type silicon, interstitial vanadium has been shown experimentally to interact with hydrogen and form at least one well documented electrically active vanadium hydrogen complex at about 0.51 eV from the conduction band [14,21].
The behaviour of this V-H complex following various thermal treatments is documented in Ref. [14]. The concentration of V-H complexes was shown to be increased upon annealing at temperatures between 75 and 150 8C, with a complete recovery of the interstitial vanadium concentration at temperatures above 175 8C. The behaviour of V-H complexes is illustrated in Fig. 3, which shows the concentration of V and V-H traps after various processing conditions, as well as their DLTS spectra. The depth profiles were obtained from an analysis of the Laplace DLTS spectra recorded with variable reverse bias and filling pulse voltages. The effect of the Debye tail was taken into account. As can be seen in Fig. 3, in the presence of hydrogen the total concentration of electrically active vanadium related complexes is reduced, indicating the formation of electrically inactive complexes involving vanadium and hydrogen. [13,14,20] along with ab initio calculations [22] strongly indicate the formation of electrically active TM-H n complexes that result from the capture of n ! 1 hydrogen atoms. Ti is a prominent example, it seems to be able to bind with up to four H atoms and become electrically inert.

Structures of TM-H complexes Depth profile studies of TM-H-related DLTS signals
Concerning the defect structure, for n ¼ 1, titanium-and vanadium-and molybdenum-hydrogen complexes form trigonal structures like the one depicted in Fig. 4, with the  metallic ion remaining close to the tetrahedral site and hydrogen located near the hexagonal site along the <111> axis [22][23][24]. For the specific case of Ti, further H attachment at equivalent sites is energetically favourable. We found that the number of hydrogen atoms that Ti is able to getter depends on the position of the Fermi-level: while in p-type material the formation of Ti-H n complexes is hindered due to electrostatic repulsion. Once we move into intrinsic and n-type Si, the binding of hydrogen to Ti becomes energetically favourable and the maximum number of H atoms that have a positive binding energy increases as the Fermi level rises in the gap. A similar behaviour was also obtained for other early metals such as vanadium or molybdenum (at least up to TM-H 2 ), which like titanium, possess deep donor levels within the band gap [25]. The increase in the number of hydrogen atoms connected to the TM ion leads to a shift or a gradual removal of the electronic states. For the Ti interstitial, a single donor is left for n ¼ 3, while full passivation is obtained for n ¼ 4.

Thermal Stability of TM-H complexes and hydrogen charge state manipulations
In both p-type and n-type Si the binding of hydrogen to the metal, in the cases we have examined, is rather weak, typically <2 eV. This implies that the hydrogenated fraction of the TMs resulting from their reactions with hydrogen is diminishingly small in Si above 200 8C. Although high temperatures are necessary to establish an adequate concentration of hydrogen throughout a silicon slice, our expectation is that the published process of carrier lifetime improvement by illumination is only significant during the cooling cycle and is in competition with other hydrogen reactions and the formation of molecular hydrogen in the silicon.
3 Lattice site changes as a route for reduced recombination While formation of complexes of transition metals and hydrogen has been shown to be effective for reducing the electrical activity of transition metals in silicon, it is also possible to render slow diffusing impurities inactive by displacing them to the substitutional site.
To date, we have shown this effect to occur for two of the three transition metals discussed so far in this paper, titanium and vanadium, when excess vacancies are introduced by implantation [26,27]. For both of these metals, displacement to the substitutional site has been observed in float zone silicon implanted with metal ions after annealing at high temperature. When comparing SIMS measurements of the total metallurgical concentration of the TM atoms to the concentration of interstitial TM's measured electrically with DLTS, a significant difference is observed between float zone grown and Czochralski grown silicon crystals after annealing.
In float zone silicon, after annealing, the electrically active fraction was seen to be smaller than the total metallurgical concentration, suggesting that the majority of the impurity atoms are not located at the interstitial site. In Cz-Si the ratio of metallurgical concentration to interstitial concentration was much closer. As well as this, redistribution of impurity atoms away from the implant site was shown to occur much more readily in Cz-Si than FZ-Si, suggesting greater diffusivity of metal atoms in this material. As no evidence of precipitation was observed in either the Cz or float zone Si samples, it was concluded that a significant fraction of TM atoms had been moved to the substitutional site in float zone silicon. At this site, these atoms have a significantly lower diffusivity, accounting for the discrepancy in redistribution between the two materials. It was also concluded that as no further evidence of electrical activity was observed either in capacitancevoltage measurements or in DLTS that the TM atoms are electrically inactive at this site [26,27].
The difference between the two materials is assumed to be primarily due to the different concentrations of available implantation induced vacancies in Cz and FZ Si. In Cz Si, vacancies are known to interact with oxygen, which is typically present at concentrations as high as 10 18 cm À3 , forming vacancy oxygen complexes, which are stable up to temperatures as high as 700 8C [28]. As the concentration of oxygen is so much higher than that of the metallic impurities, it is unlikely that interstitial impurity atoms will interact with vacancies during annealing. In contrast, in float zone silicon the concentration of oxygen is much lower, and so displacement of metal atoms to the substitutional site can occur, accounting for the differences observed in our measurements.

Conclusions
We have examined reactions between hydrogen and TMs in silicon. In p-type material we identify the lack of observations of TM-H complexes as being due to long range Coulombic repulsion between the positively charged hydrogen and the positively charged TM. However, we have demonstrated that it is possible to form FeH complexes in p-type material in the depletion region of a Schottky diode. This is attributed to the formation of the neutral charge state of the Fe i donor in the depletion region reacting with H þ . We present evidence that in our experiments (without light on the samples) the hydrogen is always in the positive charge state in the depletion region of p-type Si. Further, it is found that not all transition metals react with hydrogen under these conditions and we hypothesise that a reaction will not take place if the TM 0/þ energy level is above the Fermi level and hence retains the positive charge state in the depletion region.
We have considered the formation of TM-H complexes with multiple hydrogen atoms and conclude that in intrinsic and n-type Si, the binding of hydrogen to Ti becomes energetically favourable, and the maximum number of H atoms that have a positive binding energy increases as the Fermi level raises in the gap. A similar behaviour is also predicted for other early metals such as vanadium or molybdenum (at least up to TM-H 2 ), which like Ti, possess deep donor levels within the band gap.
Measurements of the thermal stability in all cases for the TMs show weak binding energy of H atoms of $2 eV which results in their dissociation above $200 8C. However if hydrogen is trapped in the silicon the TM-H complexes can reform on cooling.
Finally, we have investigated the behaviour of Ti and V in the presence of excess vacancies and compared the TM behavior in FZ and Cz materials. We see a reduction of electrical activity (partial passivation) and a reduction in diffusivity in FZ material when excess vacancies are present. The effect does not occur in Cz material due to the preferential reaction of the vacancies with the oxygen in Cz Si. We attribute this change of diffusivity and passivation to re-siting of the TM from interstitial to substitutional positions in the lattice.