s2fieldsofstudy: Biology Chemistry

text:
A molecular dynamics simulation study decodes the Zika virus NS5 methyltransferase bound to SAH and RNA analogue

Since 2015, widespread Zika virus outbreaks in Central and South America have caused increases in microcephaly cases, and this acute problem requires urgent attention. We employed molecular dynamics and Gaussian accelerated molecular dynamics techniques to investigate the structure of Zika NS5 protein with S-adenosyl-L-homocysteine (SAH) and an RNA analogue, namely 7-methylguanosine 5′-triphosphate (m7GTP). For the binding motif of Zika virus NS5 protein and SAH, we suggest that the four Zika NS5 substructures (residue orders: 101–112, 54–86, 127–136 and 146–161) and the residues (Ser56, Gly81, Arg84, Trp87, Thr104, Gly106, Gly107, His110, Asp146, Ile147, and Gly148) might be responsible for the selectivity of the new Zika virus drugs. For the binding motif of Zika NS5 protein and m7GTP, we suggest that the three Zika NS5 substructures (residue orders: 11–31, 146–161 and 207–218) and the residues (Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215) might be responsible for the selectivity of the new Zika virus drugs.

Currently, two major issues occur due to (1) the conformational states of protein receptors between binding and unbinding states and (2) the pathway of the inhibitor-protein interaction process.
All-atoms molecular dynamics (MD) simulations remain limited to the conformational ensembles obtained from a single long-time-scale conventional molecular dynamics (cMD) simulation due to the possible energy barriers between various intermediate states. Therefore, a multiscale simulation method that combines an enhanced sampling technique, which can take samples at various intermediate states, with an all-atoms simulation is required. Enhanced sampling techniques have been successfully applied to calculate the free-energy profiles 9 and to perform conformational sampling through accelerated molecular dynamics (aMD) 10 . These enhanced sampling methods can provide key insights into the free-energy profiles and intermediate protein structures. In general, the disadvantage of enhanced sampling techniques is the requirement to predefine protein structures, potential energies, additional energies, and reaction coordinates. Simulations using aMD or Gaussian aMD (GaMD) are enhanced sampling methods that can avoid such requirements. Through aMD, a boost of potential energy is added to the potential energy surface system resulting in a decreased energy barrier, which enables the acceleration of the transitions between low-energy states [10][11][12] . This method has been successfully applied to simulations of biological systems, and hundreds-of-nanoseconds aMD simulations can yield the same results as millisecond cMD simulations [13][14][15][16] .
In recent studies, the aMD method has produced substantial energetic noise during reweighting 17 . In aMD simulations, the applied boost potential is typically of the order of tens-to-hundreds of kilocalories per mole, which is usually greater than those of other enhanced sampling methods. Accurately reweighting aMD simulations has been problematic, especially for large protein molecules 18 . By introducing GaMD, Miao et al. provided an approach to improving the aMD method. The boost potential of the GaMD method follows a near-Gaussian distribution, for which the cumulated second-order expansion improves the reweighting of aMD simulations 19 . The reweighted free-energy profiles yielded by GaMD are in close agreement with long-time-scale cMD simulations 20 .
In the present study, the experimental biological binding affinities 21 (IC50) of SAH and 7-methylguanosine 5′-triphosphate (m7GTP) were compared using GaMD simulations; these compounds are listed in Table 1. The transfer function (ΔGbind = −RT ln(IC50)) is used to convert the IC50 values into experimental ΔGbind values, which are listed in Table 1. We applied GaMD to simulate the Zika virus NS5 protein with SAH and m7GTP. Starting from the full-length of the Zika NS5 x-ray structure, the interaction of the two molecules with the Zika virus NS5 protein was studied. We demonstrated that GaMD simulation enables a detailed analysis of the interaction of these two molecules with the Zika NS5 protein.

Theoretical Calculations Methods
Gaussian accelerated molecular dynamics. GaMD is an enhanced conformational sampling method for biomolecules that adds a harmonic boost potential to smooth the system potential energy surface 19 . When the system potential (V) is lower than a referenced energy (E), a harmonic boost potential (ΔV) is added as follows: 2 where K is a harmonic force constant. The modified system potential (V*) is given by If the system potential (V) is greater than a referenced energy (E), the harmonic boost potential (ΔV) is equal to zero. By smoothing the potential energy surface for overcoming intermedia energy barriers, the boost potential satisfies the following step. For two potential energy values V1 and V2, assume that V1 < V2 and the biased V1* < V2*. By replacing V* with equation (2), the relationship is expressed as follows: Step (1) If V1 < V2, the potential difference on the smoothed energy surface should be smaller than that of the original energy surface. By replacing V* with equation (2), the relationship is expressed as Step ( where Vmin and Vmax are the minimum and maximum potential energies. Step (3) By equation (5), we can obtain where K constant is defined as and K0 is the magnitude of the applied boost potential.
Step (4) The standard deviation (SD) of ΔV must be sufficiently small to ensure accurate reweighting 22 .
where the Vave and σ V are the average and SD of the potential energies, and σ ΔV is the SD of ΔV with σ0 as a user-specified upper limit for accurate reweighting of potential energies. In our simulations, the SDs of the total potential and dihedral potential boosts are 10 kcal/mol.
Step (5) To extend step (2), if E = Vmax, we can use equation (5) to obtain According to equations (21) and (19), K0 can be defined as: Step (6) To extend step (2), if E = Vmin + 1/k, we can use equation (8) to obtain Step (7) GaMD provides the total potential boost, dihedral potential boost, and dual potential boost in order to accelerate the molecular simulations. The boost potential (ΔV) is given as follows: where K0 is the magnitude of the applied boost potential, and Vmin and Vmax are the minimum and maximum potential energies of the system. Initially, K0 is equal to 1.0, and Vmax and Vmin are obtained through cMD simulations. The distribution and anharmonicity of the GaMD method were applied to the alanine dipeptide, chignolin, and lysozyme simulations to characterize the extent to which ΔV follows a Gaussian distribution 19 .
Reweighted free-energy calculations for GaMD simulations. The probability distribution of the selected reaction 22 coordinate A(r) is defined as P*(A), where r can be distance, angle, root-mean-square deviation, or other factors. Using the GaMD boost energies of each reaction coordinate, P*(A) can be reweighted and defined as where M is the number of bins, β is equal to the KBT, and β∆ e V r j ( ) is the ensemble-average factor of the jth bin.
For reducing the energetic noise, the ensemble-average factor can be defined as follows: According to equation (14), the first three cumulants can be defined as follows: The reweighted free energies can then be calculated by

Free-energy calculations (WHAM) for umbrella sampling simulations. A harmonic potential was
applied to the stretching 23 constraints, i.e. the distance constraints between the center of mass of the ligands and the binding pockets (Figs S1 and S7) with a force constant of 10.0 kcal/mol. The RC1 reaction coordinates follow the distance from the centre of the SAH mass to the centre of mass defined by the residues that shape the binding pocket (Gly86, Trp87, Thr104, Lys105, Asp131, Val132, Asp146, and Ile147; Fig. S1). The RC2 reaction coordinates follow the distance from the centre of the m7GTP mass to the centre of mass defined by the residues that shape the binding pocket (Lys13, Leu16, Asn17, Met19, Ser150, Ser151, and Ser215; Fig. S7). The RC1 value varied from 3.0 to 24.0 Å in 1 Å increments. The RC2 value varied from 6.0 to 22.0 Å in 1 Å increments. The MD simulations for PMF determination were performed with an initial 5-ns equilibration followed by 10-ns sampling at a given reaction coordinate value. Moreover, the umbrella sampling simulations were performed with GENESIS 1.2.0 software 24 . Then the free energy profiles (PMF) were analysed with the WHAM software 25 .
GaMD simulation of the Zika virus NS5 protein system. First, we modified a partial length of the Zika NS5 protein structure (PDB ID: 5kqs), and we used PyMOL software to modify the ligand (m7GDP to m7GTP). Second, we aligned the partial length of the Zika NS5 protein structure and the full length of the Zika NS5 protein structure (PDB ID: 5u0b). We then inserted the ligand (m7GTP) into the full-length Zika NS5 protein structure. The initial complex structures are Zika NS5 with SAH and Zika NS5 with m7GTP. These complex structures were generated and then inserted into TIP3P solvent molecules. The size of the complex structures was approximately 10.00 × 11.00 × 11.00 nm 3 . These initial complexes were then simulated using the AMBER 16 package with the AMBER FF12SB all-hydrogen amino acid and amber GAFF parameters. The geometries of the SAH and m7GTP were fully optimized, and their electrostatic potentials were obtained using a single-point calculation at the Hatree-Fock level with the 6-31 G(d,p) basis set using the GAUSSIAN 09 program 26 . Subsequently, partial charges were obtained by employing the restrained electrostatic potential procedure using the Antechamber package. All cMD simulations were performed in the isothermal-isobaric (NPT) assembly with a simulation temperature of 310 K, unless stated otherwise, by using the Verlet integrator with an integration time step of 0.002 ps and SHAKE constraints 27 for all covalent bonds involving hydrogen atoms. In the electrostatic interactions, atom-based truncation was performed using the PME 28 method, and the switch van der Waals function was used with a 2.00 nm cut-off for atom-pair lists. These complex structures were minimized for 100,000 conjugate gradient steps and then subjected to a 100-ns, isothermal, constant-volume MD simulation. Moreover, the final structures from these simulations were used in five dependent 5000-ns GaMD simulation calculations, and these structures were used in the umbrella sampling simulations.
Reweighted Free-energy calculation through potential of mean force and preresidue displacement. For the Zika NS5 protein with SAH (RC1 = 3-8 Å) and m7GTP (RC2 = 6-11 Å), all residues were obtained using preresidue displacement calculations. The preresidue displacement calculations were applied to the major barriers: RC1 = 3-8 Å and RC2 = 6-11 Å). The reaction coordinate profiles and preresidue displacement calculations were analysed using the AmberTools 16 and VMD software packages. The reaction coordinates profiles were calculated for the reaction coordinates used for the potential of mean force (PMF) calculations. The PyReweighting toolkit 22 was used to reweight the GaMD simulations for the PMF profile calculations and to examine the boost potential distributions. One-dimensional PMF profiles were also constructed using the reaction coordinates for the Zika NS5 protein with a bin size of 1.0 Å. When the number of simulation frames within a bin was lower than a certain limit, the bin was insufficiently sampled and thus was excluded for reweighting.

Results and Discussion
Potential of mean force calculation for Zika virus NS5 proteins: SAH-and m7GTP-complex GaMD simulations. Following the original paper "Reweighted free-energy calculations (PyReweighting-1D. py), " 22 we used five individual and independent 5000-ns GaMD simulations to estimate the error bar and average of the PMF, a measure of free energy, using the second-order cumulant expansion method 22 . Our results are listed in Table 1 and Fig. 1  structure-based approach was applied to identify the functionally important residues of the major energy barriers at RC1 = 3-8 and RC2 = 6-11 Å. From the complex structures at RC1 = 3-8 and RC2 = 6-11 Å, the important residues and pharmacophore regions were analysed using the Ligandscout program. Because there were many snapshots, residues with probability of >0.5 were selected for the binding mode analysis. Our results are listed in Tables 2 and 3 (Figs S1-S12 present the representative snapshots at RC1  Table 4 and Figs S13-S17. For the preresidue displacement analysis of m7GTP, the three Zika NS5 substructures (residue orders: 11-31, 146-161, and 207-218) had clear fluctuations; the results are displayed in Table 5 and Figs S18-S22.
Comparing the m7GTP binding residues of the Zika virus NS5 proteins with those of other Flavivirus NS5 proteins. The Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215 residues (PDB ID: 5U0B) were selected for comparison with other Zika NS5 proteins (PDB IDs: 5GOZ, 5GP1, 5KQR, 5KQS, 5M5B, 5TFR, 5TMH, 5ULP, 5VIM, 5WXB, 5WZ1, and 5WZ2). Our results indicated that these residues were conserved among the Zika NS5 proteins, and the results are shown in Fig. S23. The Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215 residues (PDB ID: 5U0B) were selected for comparison with the other Flavivirus NS5 proteins (PDB IDs: 3EVF (Yellow fever), 4V0R (Dengue fever), 2HKS (West Nile fever) and 4K6M (Japanese encephalitis)). The results are shown in Fig. S24. Except for two residues (Lys28 and Lys29), the residues were conserved among the NS5 proteins. The two residues of the West Nile fever, Japanese encephalitis, and Yellow fever viruses were Arg-Lys, Arg-Arg, and Lys-Arg, respectively. The Arg and Lys residues have similar structures and isoelectric points. Thus, we think that these residue differences might have a reduced impact on the binding affinity of NS65 proteins with m7GTP.

Conclusions
In this article, we used full-length Zika NS5 proteins (PDB ID: 5u0b; NS5 bound to SAH and m7GTP) as our initial structures. We employed 100-ns cMD simulations to optimize the two Zika virus NS5 protein complex structures. The RC1 reaction coordinates were defined as the distance between the centre of mass of SAH and the centre of mass of the binding pocket (Gly86, Trp87, Thr104, Lys105, Asp131, Val132, Asp146, and Ile147; Fig. S1). The RC2 reaction coordinates were defined as the distance between the centre of mass of m7GTP and the centre of mass of the binding pocket (Lys13, Leu16, Asn17, Met19, Ser150, Ser151, and Ser215). Then, we performed GaMD, preresidue displacements, and PMF calculations to predict the binding mechanisms of these molecules with Zika virus NS5 proteins. For the Zika virus NS5 protein and SAH complex, there is a major energy barrier of 8.42 ± 1.98 kcal/mol RC1 = 3-8 Å. For the Zika virus NS5 protein and m7GTP complex, there is a major energy barrier of 5.21 ± 1.21 kcal/mol at RC2 = 6-11 Å. Our energy barrier predictions were similar to the experimental data (Table 1). Moreover, we used the WHAM/umbrella sampling methods to check our reweighted free energy calculations. The energy barrier predictions were relatively close to our reweighted free-energy calculations. For the Zika NS5 protein and SAH complex, our results indicated that the four Zika NS5 substructures (residue orders: 101-112, 54-86, 127-136, and 146-161) and the Ser56, Gly81, Arg84, Trp87, Thr104, Gly106, Gly107, His110, Asp146, Ile147, and Gly148 residues affect the ability SAH to bind with the full-length Zika NS5 protein.


s2fieldsofstudy: Biology Medicine

text:
Degeneration of human photosensitive retinal ganglion cells may explain sleep and circadian rhythms disorders in Parkinson’s disease

Parkinson’s disease (PD) patients often suffer from non-motor symptoms like sleep dysregulation, mood disturbances or circadian rhythms dysfunction. The melanopsin-containing retinal ganglion cells are involved in the control and regulation of these processes and may be affected in PD, as other retinal and visual implications have been described in the disease. Number and morphology of human melanopsin-containing retinal ganglion cells were evaluated by immunohistochemistry in eyes from donors with PD or control. The Sholl number of intersections, the number of branches, and the number of terminals from the Sholl analysis were significantly reduced in PD melanopsin ganglion cells. Also, the density of these cells significantly decreased in PD compared to controls. Degeneration and impairment of the retinal melanopsin system may affect to sleep and circadian dysfunction reported in PD pathology, and its protection or stimulation may lead to better disease prospect and global quality of life of patients.

Introduction
The retina is an accessible and visible tissue, part of the central nervous system (CNS). Its well defined and highly characterized layered structure, together with the extensive knowledge about its neurons, synaptic contacts and physiology, make the retina an ideal material for pathophysiological studies of the CNS. In fact, neurodegenerative diseases mainly observed in the brain such as Parkinson's disease (PD), Alzheimer's disease, or Multiple Sclerosis present similar signs of degeneration in the retina [14], which is considered as a "window to the brain".
Intrinsically photosensitive melanopsin-containing retinal ganglion cells (mRGCs) are, together with cones and rods, retinal photoreceptors. While cones and rods are responsible for vision forming pathways, mRGCs are also in charge of the non-image forming pathways that primarily control and measure light irradiance detection [27,48]. Melanopsin, an opsin protein containing a vitamin A-based chromophore maximally sensitive at 479 nm [40,47], is the photopigment contained within mRGCs. Melanopsin-containing RGCs project to different CNS regions and regulate physiological and behavioral responses as important as circadian rhythms, pupillary reflex, melatonin production or mood [28,30].
PD is the second most common neurological disorder and affects over 10 million people worldwide (http://parkinson.org/understanding-parkinsons/causes-and-statistics). Its main motor clinical features are rigidity, tremor and bradykinesia [19,21,46], but people with PD may also have several non-motor symptoms including cognitive decline and dementia [11], gastrointestinal and cardiovascular problems [43], mood disturbance [50], visual disruption [2,55], impairment of the pupillary reflex response [54], and sleep disorders [19,46]. Sleep disorders including REM sleep behavior disorder (RBD), altered sleep, and hypersomnolence are extremely common in PD patients, affecting up to a 90% [10,56]. Moreover, people with PD also exhibit alterations in the circadian secretion pattern of melatonin [9]. Dysfunction of circadian rhythms in PD is thought to be one of the causes of sleep disturbances and it can lead to cognitive and metabolic deficits, psychiatric and mood symptoms, or cardiovascular problems, negatively impacting quality of life [56].
The defining pathological lesions of PD are Lewy bodies and associated neurites with cytoplasmic accumulation of α-synuclein phosphorylated at serine-129 (p-α-syn), and the loss of dopaminergic neurons in the substantia nigra pars compacta [5,13,19]. The latter has traditionally been considered the cause of the motor clinical manifestations. Nevertheless, PD is today mostly considered as a multisystem disorder in which other different nervous system subdivisions are affected. Brain regions involved in vision are affected in PD, including the hypothalamic suprachiasmatic nucleus [16] and the retina [6,45], both of which exhibit p-α-syn deposits. This visual system pathology in PD is accompanied by clinical findings including reduced electroretinography response and reduced visual evoked potentials, lower contrast sensitivity and impaired color and motion perception [3,39]. These all suggest that vision is strongly affected at a cellular level.
As retinal mRGCs innervate the suprachiasmatic nucleus [20] and are jointly responsible for regulating circadian rhythms, which are in turn involved in mood and sleep behaviors, mRGCs dysfunction may be at least partially involved in the PD pathological process. Others have previously proposed a link between mRGCs, circadian rhythms and sleep regulation [1,32], and a relationship between sleep disturbances and morphological impairment of mRGCs in human with aging has been described [18]. Therefore, the aim of this study was to evaluate the morphological changes of human mRGCs in PD, hypothesizing an involvement in sleep and circadian dysfunction. In this work, we show that the retinal melanopsin system is impaired in PD. We demonstrate that mRGCs degenerate in PD, as revealed by its number reduction and their morphological alterations, and this fact may be linked to the circadian and sleep disturbances suffered by PD patients.

Human retinas
Human retinas from 11 donors were obtained postmortem, within 6 h of death, from the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), the Banner Sun Health Research Institute Brain and Body Donation Program (BBDP; http://www.brainandbodydonationprogram.org/). All procedures were in accordance with the Declaration of Helsinki and with the recommendations and protocols approved by the Ethics Committee of the University of Alicante. Signed written informed consent was provided by all the participants in the study. Human donors, both men and women, were not significantly different in age, ranging from 70 to 82 years at death, and did not report any past history of retinal diseases.
The control group consisted of patients without neurodegenerative diseases (n = 5) and the Parkinson's disease group (n = 6) included subjects with a typical clinicopathological profile, diagnosed from the BBDP. Standard tests and neuropathological examinations were performed in deceased subjects as previously described [7].

Retinal histology
The human enucleated eyes were fixed in formaldehyde (3,75-4%) for 2 h at room temperature or 24-72 h at 4°C , washed in PBS and then successively cryoprotected in increasing sucrose solutions of 15%, 20% and 30%. After removing the iris, lens and vitreous body, the retina was extracted and dissected, obtaining eight quadrants. The superior-nasal portion was used for further analysis.
To determine their type and morphology, immunostained mRGCs were traced by hand in all flat-mounted retinas using a camera lucida connected to a Leica DMR microscope (Leica Microsystems). Images were then digitized, using image-editing software (Adobe Photoshop 10.0; Adobe Systems, Inc., San Jose, CA, USA). Total number of cells expressing melanopsin was counted and density of mRGCs per mm 2 was calculated.

Morphological analysis
Representative mRGCs were traced by hand in order to recreate their soma and dendritic profiles using a camera lucida (120 cells analyzed in total, 60 cells of controls and 60 of PD, 15 cells of each morphological subtype and group).
To analyze the morphology of mRGCs the Bonfire program developed in the Firestein laboratory at Rutgers University [33] was used. From digitized neuritic arbors, this software allowed us to perform a Sholl analysis and to estimate the number of branch points, the terminal neurite tips and the total number of Sholl intersections per cell [49].

Statistical analysis
Statistical analysis was performed using Prism 6 for Windows (Graphpad Software, Inc., La Jolla, CA, USA). To assess the differences of the studied variables, both globally or per mRGC subtype (M1, M1d, M2 and M3), between PD and control patients a non-parametric two-tailed Mann-Whitney test was used. Differences of the Sholl curve representing the number of intersections per distance between PD and controls were evaluated using a paired non-parametric Wilcoxon signed rank test. In all cases, a p-value lower than 0.05 was considered statistically significant.

Types of mRGCs in the human retina
In the human retina, four types of mRGCs are found. They are classified in accordance with their soma location and dendritic stratifications: M1, M1d, M2, and M3. As the diagram of Fig. 1 shows, M1, M2 and M3 cells have their soma located in the ganglion cell layer, while their dendrites stratify in different strata of the inner plexiform layer (IPL). M1 cells stratify in the S1 plexus of the IPL, M2 stratify in S5 plexus, and M3 has dendrites in both strata: S1 and S5. M1d cell is a displaced M1 cell, with its soma located in the inner nuclear layer (INL) and its dendrites in the S1 plexus of the IPL, near the INL [8]. M1d mRGC is the predominant type in the human retina, accounting for about half of all mRGCs [18,25]. An example of control and PD DAB immunostained M1d mRGC is shown in Fig. 1a and b respectively. Notice the lower dendrite complexity in PD mRGC (Fig. 1b) and its lower staining intensity when compared to controls (Fig. 1a). Identification of S1 and S5 strata can be done changing the microscope focus and using the Nomarski technique of differential interference contrast. This technique allows us to identify and differentiate the INL, the IPL, and the ganglion cell layer (GCL). Thus, S1 and S5 strata can be differentiated, without need of counterstaining, because S1 is near the INL and S5 in the opposite side of the IPL, near the GCL.

Decrease of mRGC density in PD retinas
Cell density quantification and morphological analysis were performed to evaluate differences in mRGCs between PD and control subjects. These studies were made considering the total mRGCs as well as differentiating by mRGC type. A reduction in the mRGC density and in the complexity of the melanopsin plexus was found. Fig. 2a and b are drawings representing retinal fields that show mRGCs and its plexus in control and PD. Fig. 2c and d show the density of mRGCs, expressed as number of mRGCs per mm 2 , both totally and by mRGC type. A reduction in number of mRGCs, accompanied by a drastic reduction in their plexus complexity, can be clearly seen in the drawings. The decrease in mRGC number is statistically significant (control: 4.8 ± 1.3 cells/mm 2 ; PD: 3.2 ± 0.8 cells/mm 2 ; p-value = 0.05) and it mostly affects the M1d and M2 mRGC types. In normal conditions, mRGCs make contacts to other mRGCs creating a dense plexus and have numerous dendritic beads, as the control drawing shows. In PD, the plexus is highly reduced and there are very few contacts between cells and fewer dendritic beads.

Morphological impairment of mRGCs in PD
Apart from cell density decline, morphological changes were also found in PD mRGCs. Morphology, dendritic arborization and dendritic tree size of normal and PD cells can be observed in Fig. 3. Red drawings show the elements that are placed in the IPL S5 stratum and blue represent the elements from S1 stratum, allowing the differentiation of the mRGC types. Visually, structure, size and dendritic trees are altered in PD compared to controls: dendritic area is reduced and cells have fewer and shorter ramifications. Dendritic beads, which are thought to represent synaptic contact points, are also reduced in PD (control: 44.7±25.8 beads per cell; PD: 19.3±10.9 beads per cell; p-value < 0.0001), what could be a sign of functional alteration. These morphological alterations are drastic in M1, M1d and M2 cells, while M3 cells seem to be the least affected by the disease.
Morphological changes were measured using the Sholl analysis, that includes the total number of intersections, the number of intersections per distance, and terminal points and branch points numbers. Results are shown in Fig. 4 and corroborate the differences described above.
The three analyzed measures are significantly reduced in PD, compared to controls: terminal points decrease from 16 ± 5 (controls) to 13 ± 4 (PD) (p-value < 0.001); branch points from 13± 5 to 9± 4 (p-value < 0.0001); and Sholl total number of intersections from 137,2 ± 41,3 to 106,8 ± 38,2 (p-value < 0.001). When comparing these parameters in each cell type, M1d and M2 cells show statistically significant reduced values in PD in the three measures, M1 cells show a statistically significant decrease in Sholl analysis and terminal points, and M3 were not significantly affected, although they present a tendency for fewer terminal and branch points. The Sholl analysis curve (Fig. 4g) shows the number of intersections per distance from the cell soma. Less intersections have been found in PD until a distance from the soma of 340 μm, what indicates a lower cell complexity and less ramifications of PD mRGCs, as concluded also by the less branches, terminal points, and number of total intersections that they present in comparison to controls.

Discussion
In recent years, a huge effort has been made to study the state and health of brain regions related to circadian rhythms disturbances and sleep dysregulation, like the suprachiasmatic nucleus, but these alterations are not yet completely understood and some regions are found not to be affected until advanced states of the disease [22]. The study of the retinal mRGCs is a new approach in trying to understand the cellular mechanisms underlying circadian rhythms dysfunctions in PD and may add valuable information to the current knowledge of the disease.
This study demonstrates a loss of melanopsin-immunoreactive RGCs in Parkinson's disease compared to control subjects. The density of mRGCs is significantly decreased in PD patients, and the remaining cells exhibit morphological alterations like decreased Sholl area, fewer ramifications and terminal points, and a reduced melanopsin-immunoreactive plexus. These morphological changes and numerical reduction demonstrate that mRGCs are affected in PD, probably by dying or losing melanopsin production, and it is likely that both of these would lead to functional impairment. To the best of our knowledge, this is the first study that describes alterations of mRGCs in PD.
A recent study in humans show that the mRGC density and plexus decrease with age and correlate it with the circadian rhythm dysfunction observed with aging [18]. In the present study, the mRGC type most affected is M1d, the main mRGC type in the human retina; that is also the type most affected by age [18]. M2 cells also have lower cell densities and both M1 and M2 show altered morphological parameters. These differences are not significant in the aging retina, but in Fig. 2 Representative drawings of control and PD retinal fields. Each color defines an individual mRGC. a Melanopsin plexus in a control wholemount retina. b Melanopsin plexus in a PD wholemount retina. c Total mRGC quantification (number of mRGCs per mm 2 ) and comparison between control and PD subjects. d Comparison of the mRGC density per cell type in control and PD subjects. Scale bar, 100 μm. Data is presented as mean ± s.d. *P < 0.05, **P < 0.01 PD it seems that almost all mRGCs show morphological abnormalities as well as a numerical decline. As mRGCs innervate the suprachiasmatic nucleus [20], it is expected that these morphological alterations lead to a dysfunction mostly related with circadian rhythms, mood, and sleep; and also with the pupillary reflex: the major mRGC functions. Morphological and connectivity studies about mRGCs have also demonstrated its relationship with dopaminergic cells, which make contacts in the S1 strata of the IPL with mRGC somas and dendrites, mainly with Fig. 3 Representative drawings of control and PD mRGCs. Blue indicates the parts of the cell located in the S1 IPL layer and red shows the parts of the cell located in the S5 IPL layer. a, b M1 mRGC in control and PD respectively. c, d M1d mRGC in control and PD respectively. e, f M2 mRGC in control and PD respectively. g, h M3 mRGC in control and PD respectively. Scale bar, 100 μm the M1d type [38]. Diminution of dopamine levels in the retina in PD [26] may be one of the causes of M1d cell degeneration, as this would represent a loss of one of their main synaptic inputs.
In animal models, MPTP-treated monkeys exhibit dopaminergic system impairment and circadian rhythm disruption with altered sleep/wake cycle, REM sleep impairment and daytime sleepiness [15,23,51,56]. Also, in P23H blind rats, degeneration of mRGCs statistically correlates with circadian rhythms impairment [34]. Other existing works that analyzed the effect of parkinsonism in circadian rhythms described changes in the expression of the "clock genes" [12], in circadian melatonin secretion [9], in pupillary reflex [4,54], depression [57] and in REM sleep [10,52], all directly or indirectly controlled and affected by mRGCs. But the Sholl area comparison of PD and control mRGCs globally (e) and per cell type (f). Data is presented as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. g Sholl analysis curve representing number of intersections per distance from soma, comparing total controls and PD mRGCs. Data is presented as mean ± s.e.m. suprachiasmatic nucleus has been found to be unaffected until advanced stages of the disease, suggesting that there are other components of the circadian system causing circadian abnormalities in PD [22]. Thus, it is easy to question the implication of the retina, and specifically of the mRGCs, in circadian dysfunction in PD, but until now no cellular studies were available to determine its real contribution. The retinal melanopsin system abnormalities detected in PD in the present study help to explain some of the circadian and sleep problems that are common in the disease, as it probably contributes to or worsens them. The loss of mRGCs have also been described in other neurological pathologies like Alzheimer's disease and diabetic retinopathy [31,44] where its impairment is related to circadian rhythm alterations and sleep disorders [32,35].
There is growing evidence that circadian rhythm disorders, normally accompanied by sleep disruption, not only negatively affect the patients' quality of life but may also accelerate the progression of neurodegenerative disease pathology [41]. The identification and management of these symptoms is therefore important not only for a clinical benefit but perhaps also for modulation of disease progression. In this sense, knowing the effect that the retina and mRGCs may have in the progression of circadian disorders, eye protection should be recommended to patients. Additionally, novel therapies using light stimulation to synchronize circadian rhythms are demonstrating beneficial results in PD [24,29,36]. Martino et al. found that long-term light therapy improves sleep quality, reduces awakenings during the night and increases the total sleep time [42]. Light therapy was also found to be effective for excessive daytime sleepiness and global sleep quality [53]. A cellular explanation of this light therapy success might possibly invoke the stimulation of mRGCs, leading to dopamine release and to circadian rhythms synchronization, globally improving PD pathology [37].

Conclusions
In summary, the present work demonstrates that the retinal melanopsin system is affected in PD. This fact has clinical implications for PD-related circadian rhythm alteration as well as for mood and sleep disorders. Protecting the retina to prolong mRGC health and using light therapies could be beneficial for the maintenance of circadian rhythms and for improving global life quality of patients.


extfieldsofstudy: Chemistry Medicine

text:
Development of podophyllotoxin-loaded nanostructured lipid carriers for the treatment of condyloma acuminatum

Condyloma acuminatum (CA) is a common sexually transmitted disease caused by human papillomavirus (HPV). Podophyllotoxin (POD), a cytotoxic compound, is able to effectively treat HPV; however, the severe irritation side effects of POD restrict its use as a treatment for CA. The aim of the present study was to construct novel POD-loaded nanostructured nanolipid carriers (POD-NLCs) and evaluate their physicochemical characteristics and cytotoxicity. POD-NLCs (0.5%) were prepared using emulsion-evaporation and low temperature-solidification methods with optimized conditions and preparations. Subsequently, the POD-NLCs were physicochemically characterized and their in vitro and in vivo release efficiencies and in vitro cytotoxicity were studied. The prepared POD-NLCs had an average particle size, ζ potential, polydispersity index and encapsulation efficacy of 178.5±20 nm, −27±0.5 mV, 0.18±0.01 and 82.9±2%, respectively. In vitro and in vivo release studies demonstrated that POD-NLCs are able to provide sustained drug delivery for 72 h in vitro and 10 h in the mucosa. Compared with a tincture formulation of POD (POD-T), POD-NLC induced less inflammatory cytokine production in the cervical mucous and led to a decreased histopathological score. In addition, a cytotoxicity assay demonstrated that inhibition of the POD-NLCs was 98.4% at 24 h and remained >98% up to 72 h. Furthermore, more cells were arrested in the G2/M phase of the cell cycle following POD-NLC treatment compared with the POD-T treatment. The present study provides evidence that POD-NLC is a promising delivery system for the treatment of CA.

Introduction
Condyloma acuminatum (CA), also known as genital warts, is a common sexually transmitted disease that causes prolific lesions on the genitalia, perianal area, vagina and cervix; CA is caused by certain types of human papillomavirus (HPV) (1). Nearly all sexually active individuals become infected with HPV at least once during their lifetime (2). In females, HPV infection can result in cervical and vaginal intraepithelial neoplasia (3,4). Therefore, the prevention and treatment of HPV infection is a public health issue.
Podophyllotoxin (POD), a cytotoxic compound extracted from the roots and rhizomes of Berberidaceae, is primarily used as an anti-tumor and anti-viral drug (5). POD tincture (POD-T; 0.5%) is commonly used as a first-line agent for the treatment of CA (5). However, the tincture is prepared in alcohol, which can cause severe irritation to the vagina, cervix and other mucosae, limiting its application (6). In addition, epidermal uptake and targeting of POD-T are poor and systemic absorption leads to severe side effects, including gastrointestinal disturbances, thrombocytopenia, leukopenia, abnormal liver function, ataxia and peripheral nerve palsy (7,8). To overcome the side effects of POD-T application on mucosae, it is necessary to develop novel alternative formulations to prolong the local residence time in cervical epithelial cells infected by HPV and reduce cervical irritation and systemic toxicity.
At present, localized delivery of chemotherapeutic drugs to the cervix is used for a number of reasons, including to treat vaginal infections, as contraception and as HIV prevention, with various formulations including gels, creams, suppositories, films and nanoparticle drug carriers (NDCs) available (9). A previous study reported that NDCs improve the physical and chemical properties of drugs, provide sustained, targeted release and reduce systemic absorption (10). Nanostructured lipid carriers (NLCs) are a novel type of NDC developed from solid lipid nanoparticles, in which a solid matrix is blended with a fluid lipid to improve drug loading capacity and release properties (10)(11)(12). NLCs are able to adhere to the skin surface and transport drugs in a controlled manner, while causing minimal irritation (13).
In the present study, a POD-NLC formula containing 5 mg/ml POD was prepared and optimized. Tibetan miniature pigs were selected as an experimental animal model to investigate the absorption and metabolism of POD-NLCs and the mucosal irritation caused by POD-NLCs in the porcine cervix. The physicochemical characteristics of this formula, including particle size, entrapment efficiency and polydispersibility, were evaluated to 0.0005, 0.005, 0.05, 0.5 and 5 µg/ml with sterile water. The stability of POD-NLCs was investigated at 4˚C and at room temperature (25˚C). The release characteristics were investigated in vitro and in vivo, and cervical mucosal tissue irritation was investigated in vivo. The cytotoxicity of POD-NLCs on VK2/E6E7 cells and cell cycle arrest were also investigated.
POD loaded NLC preparation. A POD-NLC formula containing 5 mg/ml [POD-NLC (0.5%)] was prepared using the emulsion-evaporation and low temperature-solidification methods, as previously described (14). The formulation was optimized using orthogonal array testing. Briefly, 50 mg of POD, glyceryl monostearate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and octyl/decyl acid triglyceride (Sigma; Merck KGaA) were dissolved in methylene chloride and lecithin (Sigma; Merck KGaA) was dissolved by sonication in ethanol (≥99.7%) for 30 min at 4˚C. Methylene chloride and ethanol solutions were mixed to form an organic phase. Subsequently, the organic phase was rapidly injected into a poloxamer aqueous solution, which was subsequently stirred (245 x g) in a water bath at 80˚C (DF-101S; Xingshuo Instrument, Guangzhou, China) for 3-4 h. Subsequently, a coarse emulsion was rapidly mixed with 5 ml of water and ice mixture and sonicated (245 x g) at 4˚C for 30 min.
Characterization of POD-NLC. The average particle size, ζ potential and polydispersity index (PDI) were measured using a Malvern Zetasizer 3000HSA (Malvern Instruments, Ltd., Malvern, UK). All samples were prepared in triplicate. The morphology of the POD-NLCs was observed using transmission electron microscopy (TEM; Tecnai G2 Spirit T12; FEI; Thermo Fisher Scientific, Inc., Waltham, MA, USA). To prepare the sample for TEM examination, 50 µl POD-NSC was added into 5 ml ethanol. Following ultrasonic dispersion for 15 min, the dispersion was repeatedly scooped with a carbon film copper net sample for 5 min. Then, the dispersion was oven-dried at 60˚C. The prepared samples was used for TEM examination as follows: accelerating voltage, 120 kV; dot resolution, 0.34 nm; line resolution, 0.20 nm; magnification, x50-800,000.
Entrapment efficiency (EE) analysis. EE analysis was carried out as previously described (15). The nanoparticles were isolated using ultrafiltration filters (0.22 um) and centrifuged for 30 min at 23,128 x g. W free is the amount of soluble free drug in the supernatant and W total is the amount of drug added to the emulsion. The samples were analyzed in triplicate and assessed using high-performance liquid chromatography (HPLC, without internal standards; LC-20A; Shimadzu Corporation, Kyoto, Japan) with a Ecosil C18 analytical column (4.6x250 mm; 5 µm) was used and maintained at 40˚C. The mobile phase contained methanol and glacial acetic acid (52:48, v/v; flow rate, 1 ml/min). The injection volume was 20 ml, the retention time was approximately 6 min and the detection wavelength was 292 nm. The recovery rate ranged from 98.6 to 99.6%. The POD concentration, with a range of 1-500 µg/ml (r=correlation coefficient, 0.9995) was measured to detect the POD formulations. The EE was calculated using the following formula: EE (%) = [(W total -W free )/W total ] x 100.
In vitro release studies. The release kinetics of POD-NLC were studied using Franz diffusion cells. Simulated vaginal fluid (SVF) was used as the release medium and was prepared according to a previously described method (16). Briefly, 0.5 ml tested solutions were added into donor chambers and filtered through the cellulose acetate membrane (0.45 µm; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and aliquots (0.1 ml) were subsequently withdrawn from receptor chambers at 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84 and 96 h. Samples were diluted with methanol (99.9 %) six times and assessed by HPLC.
Animal model. A total of 15 female Tibetan mini-pigs (8 months old; 25 kg) were purchased from the Center of Experimental Animals, Southern Medical University (Guangzhou, China). The pigs were maintained in large hog pens (n=7 per hog pen) in a natural environment (25˚C, 12 h dark cycle) with free access to food and water. All the experimental animals and the protocol (permit number for pigs: 44002100008963) used in the present study were approved by the Experimental Animal Ethics Committee of the Southern Medical University (Guangzhou, China).
In vivo drug release studies in cervical mucosal tissue and cervical mucus. For the purpose of dynamics analysis, 15 female Tibetan mini-pigs were randomly divided into four subgroups and administrated with 1, 5 and 20 mg/ml POD-T, and 5 mg/ml POD-NLC. Cervical secretions were extracted from ophthalmic sponges using a previously described method (17); 1, 5 and 20 mg/ml POD-T, and 5 mg/ml POD-NLC (0.5 ml) were administrated on the cervix for one treatment. Cervical mucus was collected at 2, 4, 6, 8 and 10 h using sponges held in the cervix and left to stand for 5 min as previously described (18). All sponges were transferred immediately into sterile tubes and stored at -20˚C. Following anesthesia, cervical mucosal tissue was collected by means of a simple punch biopsy as previously described method (19) at 2, 4, 6, 8, 10 h following administration of 1, 5 and 20 mg/ml POD-T, and 5 mg/ml POD-NLC (0.5 ml) as previous stated, and the coated cervical mucosa determined to was 3.14 cm 2 . Subsequently, tissues were rinsed with normal saline and stored at -20˚C. POD was extracted from the sponge and cervical tissue by homogenization in 1.0 ml methanol followed by centrifugation (24,573 x g for 15 min at 4˚C).

Evaluation of inhibition of proliferation. POD-NLC-induced
inhibition of cell proliferation was tested using VK2/E6E7 cells. Following attaching for 24 h, cells were exposed to a series of doses (0.0005, 0.005, 0.05, 0.5 and 5 mg/ml) of POD-NLC or POD, which was diluted with sterile water. The control group contained only the culture medium. Following incubation for 24, 48 and 72 h, 10 µl Cell Counting kit-8 (CCK-8) solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well of the plate followed by incubation at 37˚C for 4 h. The absorbance was measured at a wavelength of 450 nm using a Microplate Luminometer (Turner Designs, Sunnyvale, CA, USA). All experiments were repeated three times. The proliferation inhibition ratio of cells was calculated using the following formula: Inhibition ratio = [1 -A(POD or POD-NLC group)/A(control group)] x 100%, where A is the absorbance value at a wavelength of 450 nm.
Apoptosis assay. Cell apoptosis was detected using the Annexin V-fluorescein isothiocyanate (FITC) kit (containing binding buffer, Annexin V-FITC and propidium iodide; BD Biosciences, Franklin Lakes, NJ, USA) according to a previously described method (23). Briefly, 1x10 6 VK2/E6E7 cells were incubated with POD or POD-NLC at a dose of 0.05 µg/ml for 48 h. Cells were subsequently harvested and re-suspended in PBS. The cells were counted and 1.5x10 5 cells were re-suspended in 200 µl binding buffer and 5 µl Annexin V-FITC, and stained for 10 min at room temperature. The cells were centrifuged (120 x g for 5 min at 28˚C) and re-suspended with 200 µl binding buffer and then added 10 µl propidium iodide (PI) and stained for 5 min on ice. Finally, the stained cells were analyzed using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA). Data were analyzed with NovoExpress software (version 1.2.4; ACEA Biosciences).
Cell cycle analysis. VK2/E6E7 cells (1x10 6 ) incubated with POD or POD-NLC at a dose of 0.05 µg/ml for 48 h were harvested by centrifuged (120 x g for 5 min at 28˚C) then washed twice with cold PBS. Following fixing with 75% ethanol for 24 h at 4˚C, the cells were washed twice with PBS and stained with 300 µl PI (100 g/ml) for 30 min at 4˚C in the dark. The cell cycle was analyzed using a NovoExpress software (version 1. Statistical analysis. The experimental groups were compared with the control groups via one-way analysis of variance with repeated measures and the Fisher's Least Significant Difference. P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± standard error of the mean. All calculations were performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA).

Characterization of POD-NLC.
In the present study, 0.5% POD-NLC was prepared using emulsion-evaporation and low temperature-solidification methods. The POD-NLCs were spherical or ellipsoidal (Fig. 1A) and the mean particle size was 178.5±20 nm (Fig. 1B) with a PDI of 0.18±0.01 (data not shown), which implies that the prepared POD-NLCs were monodisperse. The ζ potential was -27±0.5 mV (Fig. 1C), and the pH was 6.20±0.04 (Table I). The preparation's entrapment efficiency were calculated using the following formula: EE (%) = [(W total -W free )/W total ] x 100. The POD entrapment efficiency in the nanoparticles was determined to be 82.9±2% by HPLC (Table I). The final results showed POD-NLC (5 mg/ml) has a really high entrapment efficiency. The POD-NLC formulation was stable when stored at 4˚C for 6 months or at room temperature for 3 months, with no stratification, sedimentation or POD crystallization observed.
POD-NLCs sustain drug release and extend the local action time in vitro and in vivo. To investigate the release of POD-NLCs in vitro, the medium was collected from Franz diffusion cells following POD-NLC and POD-T treatment and analyzed using HPLC. At 6 h following treatment, nearly 69% of POD was released from POD-T, compared with 8.8% from POD-NLCs ( Fig. 2A). At 24 h following treatment, 30.98% of the encapsulated POD was released from POD-NLCs and 64.90% was released following 72 h ( Fig. 2A). Furthermore, in vivo release studies were performed with cervical mucus and mucosal tissues. The results demonstrated that the concentration of POD in cervical mucus, released from the POD-NLC (5 mg/ml) formulation, decreased gradually following administration but increased compared with the POD-T (5 mg/ml) group at each time point and increased compared with the POD-T (20 mg/ml) group at 4 h (115.3 vs. 95.8 µg/ml, respectively; P<0.05). The POD-NLC group maintained an increased concentration at 10 h compared with POD-NLC group (189.6 vs. 34.2 µg/ml; Fig. 2B). In the cervical mucosal tissue, POD-T administrations at different concentrations (1, 5 and 20 mg/ml) reached the maximum concentration of POD 4 h following administration, followed by a decline. In the 5 mg/ml POD-NLC group, however, the POD concentration in the cervical mucosal tissue continued to increase up to 8 h following administration. At 10 h following administration, the POD concentration in mucosal tissues was not markedly elevated in the 5 mg/ml POD-NLC group compared with the 5 mg/ml POD-T group (Fig. 2C). These results demonstrated that, compared with POD-T administration, the POD-NLC exhibited a more sustained release of POD, which may extend the local action time in vitro and in vivo.

POD-NLC prevents injury and inflammatory cytokine production in the cervical mucosal tissue compared with POD-T.
Following administration of the formulations, damage to the cervical mucosa was assessed for 72 h. In the low-dose POD-NLC group (0.5 ml/day), the cervical mucosal epithelium consisted of 5-10 layers of cells with structural integrity. Ulcers and hemorrhaging with neutrophil and mononuclear cell infiltrations were observed in the high-dose POD-NLC and POD-T groups. In addition, necrosis of the mucosal epithelium was observed in the POD-T group (Fig. 3A). In the lamina propria of the cervical mucosal tissue, no inflammatory infiltration was observed in the low-dose POD-NLC group (Fig. 3A-g), whereas a large number of infiltrating neutrophils and mononuclear cells were observed in the high-dose POD-NLC and POD-T groups (Fig. 3A-i and -j, respectively). Furthermore, submucosal vessels in the low-dose POD-NLC group appeared normal (Fig. 3A-l); however, in the high-dose POD-NLC group, capillary congestion, hemorrhaging and infiltration of mononuclear cells into the perivascular region were observed. In the POD-T group, thrombosis of partial vessels occurred (Fig. 3A-o). Histopathological scores were calculated by assessing the results in multiple regions of the cervical mucosal tissue. Histopathological scores of the low-dose and medium-dose POD-NLC group were significantly lower compared with the POD-T group (4.3±0.8 vs. 29.3±29.3; P<0.05). No significant difference was observed between the low-dose POD-NLC and saline groups (4.3±0.8 vs. 3.3±1.0; P>0.05; Fig. 3B). Furthermore, the levels of IL-6, -8 and -1β were significantly lower in the low-dose POD-NLC group compared with the high-dose group (P<0.01), and were not significantly different compared with the NS group (Fig. 3C-E). The levels of inflammatory cytokines in the POD-T group were significantly increased compared with the low-medium-and high-dose POD-NLC groups (Fig. 3C-E). Taken together, these results demonstrate that POD-NLCs cause less injury and inflammatory cytokine production in the cervical mucosal tissue compared with POD-T.

POD-NLC inhibits the proliferation of VK2/E6E7 cells.
To evaluate the CA-inhibitory activity of POD-NLCs, the effect of POD-NLC on the proliferation of VK2/E6E7 cells was investigated. Compared with POD, POD-NLC resulted in decreased cell viability at high concentration (0.5 and 5 µg/ml). The proliferation inhibition rate of VK2/E6E7 cells treated with POD-NLC reached 99% at a dose of 5 µg/ml (Fig. 4A). However, the inhibition of POD was 64.5% at a dose of 5 µg/ml and did not increase further with prolonged exposure time (Fig. 4B). The inhibition of POD-NLCs reached 98.4% at 24 h and persisted at >98% from 24 to 72 h (Fig. 4B), which is increased compared with the POD treatment group.
Hoechst 33342 staining was performed to assess the morphology of the nuclei (Fig. 4C). It was determined that POD-NLC-treated VK2/E6E7 cells had apoptotic morphology, including cell membrane shrinkage, cell shedding and chromatin condensation. POD-treated cells exhibited a similar pattern (Fig. 4C). In the POD-NLC-and POD-treated groups, the apoptosis rates increased by 13.2 and 7.9%, respectively, compared with the control group (Fig. 4D). Cell cycle distribution was analyzed using flow cytometry. Following treatment for 48 h, VK2/E6E7 cells in the POD-and POD-NLC-treated groups were predominantly in the G2/M phase, and fewer cells were observed in the G0/G1 phase (Fig. 4E). POD-NLC treatment induced G2/M arrest to a greater extent compared with the POD treatment (0.863±0.085 vs. 0.602±0.043, respectively; P<0.01; Fig. 4E). Collectively, these results demonstrate that POD-NLC has a more prominent effect on VK2/E6E7 cell viability compared with the POD treatment.

Discussion
POD-T (0.5%) has been reported to be an effective initial drug for the treatment of CA (24). However, it has been demonstrated to cause severe irritation when applied to the vagina, cervix or other mucosae (25). Recently, POD-NLCs have been proposed as an attractive delivery system for the treatment of HPV and associated diseases with low systemic adverse effects (14,15). Previous studies have reported that nanoparticles of small diameter (<100 nm) would be more likely to be hindered or immobilized by cervical mucus compared with nanoparticles of larger diameter (200 nm) (26,27). In the present study,   0.5% POD-NLCs were prepared with an average diameter of 178.5±20 nm, which is within the optimal range for mucosal drug delivery. The mucosae of the reproductive tract secrete appropriate amounts of mucus, mainly composed of mucins, which are responsible for mucoadhesion and elasticity (28). Ramineni et al (29) proposed that the characteristics of mucus could cause it to act as a barrier limiting drug penetration. Additionally, the retention time of traditional formulations (including solutions, suspensions and emulsions) in the reproductive tract surface is insufficient, making it difficult to achieve the desired therapeutic effect (30). Nanoparticles with mucoadhesive properties have previously been used to facilitate drug administration to the mucosa and improve the active effects of transmucosal administration (31). In the present study, in vitro experimentation indicated that the residence time of POD-NLCs was increased compared with POD-T. POD-NLCs enable sustained drug delivery for 60 h, consistent with results of a previous study, which used POD-NLCs for targeted drug delivery (12). In the present study, the in vivo drug release from POD-NLCs in Tibetan miniature pigs was monitored by detecting the amount of POD in the cervical mucus and mucosal tissue. The results demonstrated that the concentration of POD in the mucus of the POD-T group decreased rapidly following transmucosal administration. The amount of POD in the mucus and the mucosal tissue was increased in the POD-NLC group compared with the POD-T group. This demonstrates that, although POD-T penetrates the mucus and mucosa more rapidly, POD-NLCs are able to persist in the cervical mucus and on the mucosal surface and exhibit sustained-release for 10 h and controlled-release properties as high levels of POD were maintained. These results suggest that POD-NLCs possesses mucoadhesive abilities and provides controlled drug release, which is more suitable for the treatment of CA through the cervical mucosal tissue compared with POD-T.
Previous reports have demonstrated that certain antimicrobial agents used in the female reproductive tract can cause damage to the mucosal barrier; drugs that alter the reproductive tract mucosal tissue environment and disrupt this barrier can increase the risk of infection by sexually transmitted pathogens, including human immunodeficiency virus (HIV), HPV and Chlamydia trachomatis (32)(33)(34). When the cervical mucosal tissue is stimulated, induced epithelial cell hyperactivity leads to the release of a series of chemokines and cytokines, eliciting an immune response through the transmission of signals to submucosal cells and tissues (24). Cytokines, including IL-1β, IL-8 and IL-6, are used as markers for the potential toxicity of drugs administered to the reproductive tract mucosa (20). NLCs can be prepared using both solid and liquid lipids, which induce minimal skin irritation (35). In the present study, the irritation caused to the cervical mucosal tissue of Tibetan mini pigs following repeated administrations of different doses of POD-NLC was evaluated. In the POD-T and high-dose and medium-dose POD-NLC-treated groups, severe destruction to the cervical mucosal tissue and elevated levels of IL-1β, -6 and -8 were observed, suggesting induction of inflammatory responses. However, the low-dose POD-NLC group did not result in any apparent pathological alteration in the mucosal epithelium. Therefore, POD-NLCs can maintain the integrity of the cervical mucosal epithelial barrier whilst also preventing an increase in the release of pro-inflammatory cytokines. The results of the present study are in agreement with previous studies, which demonstrated that the intermittent use of small doses of POD-NLCs is an efficient therapeutic method for preventing irritation of the cervical mucosal tissue (36).
E6 and E7 are oncoproteins encoded by HPV that transform cells by cooperatively targeting diverse cellular pathways involved in the regulation of cell cycle control, apoptosis and cell polarity control networks (37). Therefore, the HPV-transformed VK2/E6E7 cell line was used to investigate the effects of POD-NLC treatment on the viability of cells. Cytotoxic effects of POD were previously reported to influence different stages of cell cycle, including inhibition of RNA and DNA synthesis and polymerization, and prevention of cell division through inhibition of microtubule assembly (38). In a previously published study, the authors of the present study reported that POD increases the intracellular calcium concentration and upregulates the expression of 78 kDa glucose-regulated protein homolog, glucose requlated protein 94 and calpain2 mRNA in VK2/E6E7 cells (19). To determine the effect of POD-NLCs on VK2/E6E7 cells, proliferation inhibition rate and cytotoxicity studies were performed. The results of the present study demonstrated that more efficient inhibition of VK2/E6E7 cell proliferation was achieved following treatment with POD-NLCs compared with the POD group. POD-NLC treatment induced a proliferation inhibition rate of >99% compared with 68.9% achieved by POD treatment. The elevated proliferation inhibition rate induced by POD-NLC treatment may be due to increased uptake of POD into VK2/E6E7 cells. In addition, compared with the POD treatment, POD-NLC resulted in increased proportion of cells in the G2/M stage and elevated apoptosis of VK2/E6E7 cells. Although the mechanism underlying these results remains to be elucidated, the observations indicate that POD-NLCs demonstrated more effective anti-HPV activity compared with free POD.
In conclusion, POD-NLCs demonstrate favorable physicochemical characteristics for transmucosal delivery, providing sustained release and controlled delivery in the cervical mucosal tissue. POD-NLCs can inhibit the growth of VK2/E6E7 cells via G2/M arrest and inhibit cell proliferation more efficiently compared with the POD. The present study demonstrated that NLCs is a promising delivery system for POD in the treatment of CA.


extfieldsofstudy: Biology Medicine

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Immuno-impedimetric Biosensor for Onsite Monitoring of Ascospores and Forecasting of Sclerotinia Stem Rot of Canola

Sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is a destructive disease of canola and many other broadleaf crops. The primary inoculum responsible for initiating Sclerotinia epidemics is airborne ascospores released from the apothecia of sclerotia. Timely detection of the presence of airborne ascospores can serve as an early-warning system for forecasting and management of the disease. A major challenge is to develop a portable and automated device which can be deployed onsite to detect and quantify the presence of minute quantities of ascospores in the air and serves as a unit in a network of systems for forecasting of the epidemic. In this communication, we present the development of an impedimetric non-Faradaic biosensor based on anti-S. sclerotiorum polyclonal antibodies as probes to selectively capture the ascospores and sense their binding by an impedance based interdigitated electrode which was found to directly and unambiguously correlate the number of ascospores on sensor surface with the impedance response.

SCIeNtIfIC REPORTS | (2018) 8:12396 | DOI: 10.1038/s41598-018-30167-5 the appearance of symptoms in the crops. Systematic application of fungicides is unprofitable because the outbreak of sclerotinia incidence can vary greatly among fields and years.
Therefore, a number of forecasting systems have been developed to predict the risk of stem rot infection 8,9 . The methodology adopted for risk assessment includes recording the amount of continuous rainfall for a number of days, soil moisture and apothecium development, temperature, crop canopy development, crop rotation, and crop disease levels in the previous years. Additional tools such as testing petals for ascospores infection and weather-based forecasting maps have been adopted in Canada 10,11 . However, these approaches for risk assessments are time consuming, labor intensive as they require constant field testing, and yet may not predict the risk in a timely manner.
As airborne ascospores are the dominant source of the spread of stem rot infection in susceptible plants, methods that can directly detect airborne ascospores offer the best and most direct measure of the risk of crop infection. In the case of carrots 12 , where a detailed correlation between the presence of airborne ascospores and incidence of Sclerotinia rot carrot epidemic are available, the initial occurrence of the disease was observed 8 and 34 days following the detection of 9.5 and 2 ascospores per m 3 of air, respectively. In recent years, quantitative real-time polymerase chain reaction (qPCR) has been developed as the method of choice for monitoring airborne ascospores by amplifying a selected segment of their DNA for detection and quantification [12][13][14][15][16][17] . Although qPCR has the sensitivity and selectivity to detect the presence of pathogens to a level as low as a single ascospore in the sample, it has a number of disadvantages in terms of cost and complexity of the method due to the simultaneous requirements of thermal cycling and fluorescence detection which renders the technique unsuitable for routine onsite field applications.
In this article, we report on the design and development of a biosensor based on anti-S. sclerotiorum antibodies as probes immobilized on interdigitated electrodes (IDEs) and sense the binding of the ascospores by label-free non-Faradaic impedimetric detection for sensitive and selective detection and quantification of ascospores. As devices based on label-free non-Faradaic impedance detection are amenable to microfabrication and miniaturization, our goal is to develop a low cost, miniaturized, and automated biosensor for onsite monitoring of the number of ascospores suspended in the air around the canola fields and serves as an early warning system for farmers to forecast and manage the outbreak of Sclerotinia stem rot epidemic of canola.

Results and Discussion
Antibody immobilization. One of the most important factors contributing to the development of a sensitive antibody-based biosensor is the ability to immobilize a high density of oriented antibodies covalently on the surface of the interdigitated electrodes (IDEs) so that the paratopes are free in the solution and available for efficient binding with the target antigens [18][19][20] . Sensitivity and stability, achieved in biosensors with well oriented covalently immobilized antibodies can be two orders of magnitude higher than biosensors with randomly oriented antibodies [21][22][23] . Figure 1 illustrates the surface functionalization steps and the process used to achieve oriented immobilization of anti-S. sclerotiorum antibodies on the surface of the IDEs. The first step in the functionalization of the gold surface of the IDE is the formation of a self-assembled monolayer (SAM) of alkanethiols with distal carboxylic acid end group. Alkanethiols with different chain lengths, viz 1-Mercapto-11-undecanoic acid (11-MUA) and 1-Mercapto-6-hexanoic acid (6-MHA) in aqueous ethanol solution with a molar ratio of 1:10 were used to form SAM to reduce steric crowding of the surface carboxylic acid end group. Treatment of the surface with EDC/NHS solution activates the carboxylic acid group by the formation of NHS ester which can efficiently react with the amino group of APBA to form an IDE with the gold electrode surface functionalized with boronic acid group.
Cyclic boronate esters are formed when boronic acid reacts with the 1,2-or 1,3-diol group of the carbohydrate moiety present in the fragment crystalizable region (Fc) of the antibody 18,21,24,25 . As the Fc region is located far away from the antibody binding sites and the boronate ester formation is specific to the carbohydrate moiety, the immobilization reaction afforded by boronic acid functionalized IDE provides well oriented antibodies with their paratopes facing the solution and readily available for efficient binding with the target antigens in the solution.

Impedance spectra measurements and ascospores quantification. Electrochemical Impedance
Spectroscopy is a powerful technique for investigating electrode/electrolyte interface as it provides rich information about the interface, interface structure, and interfacial electrode processes [26][27][28][29][30][31][32][33][34] . An impedance spectrum is obtained by applying a current or voltage excitation perturbation in an electrochemical cell and measuring the voltage or the current response as a function of the applied excitation frequency. Impedance is a measure of the opposition to the flow of current, arising from ion diffusion, electrode kinetics, redox reactions, and molecular interactions at the electrode surface, when an alternating excitation voltage is applied to the cell.
Depending on the presence or absence of redox active chemicals in the solution used for impedance measurement, a biosensor can be classified as either Faradaic or non-Faradaic [26][27][28][29][30][31][32][33][34] . In the Faradaic based biosensor, the factors contributing to the impedance are electrolyte resistance, double layer capacitance, interfacial electron transfer, and Warburg impedance. Whereas, in the non-Faradaic based biosensor such as the immuno-impedimetric biosensor described here, the contributions from the electrolyte resistance and the interfacial capacitances dominate the impedance of the system. In a non-Faradaic biosensor, the interfacial capacitances are sensitive to the probe-analyte binding occurring on the surface of the electrode and can be used for detection and quantification of target antigens. The total capacitance (C tot ) of a sensor electrode can be considered as a combination of capacitances attributed to the SAM (C SAM ), recognition layer (C REC ), and double layer (C DL ) connected in series [26][27][28][29][30][31][32][33][34] and the impedance is related to capacitance as, where, ω = 2πf, is the angular frequency and f is the applied frequency in hertz.
Due to the reciprocal nature of the relation, the total capacitance of the electrode/electrolyte interface is most sensitive to the changes in the magnitude of the smallest capacitance in the series. As C DL in aqueous solution is normally very large value 35,36 , on the order of µF/cm 2 , a sensitive non-Faradaic impedance biosensor should be designed such that the C SAM is as large as possible, and C REC as small as possible [26][27][28][29][30][31][32][33][34] . Therefore, surface modification and immobilization of the antibodies play critical role in the development of a sensitive impedance based non-Faradaic biosensors. Figure 2 shows the impedance spectra, the plots of the impedance magnitude and phase versus frequency, of the applied sinusoidal excitation potential (10 mV at 0 V DC) obtained for the IDE before modification (bare gold), after modification with SAM, and subsequently after immobilization of anti-S. sclerotiorum antibodies. It is known that the impedance magnitude of a non-faradaic impedance spectrum is dominated by interface capacitance, solvent resistance, and dielectric capacitance in the low, intermediate, and high frequency region of the spectrum, respectively 37,38 . As presented in Fig. 2, these regions of the spectrum are observed in the impedance spectra as expected. It is important to note that the capacitance of the system, as expected, is better represented by a constant phase element as indicated by the magnitude of the phase angle in the low frequency region. As shown in Fig. 2, modification of the bare gold IDE by SAM led to a huge increase in the impedance, as expected for capacitances connected in series with the double layer, because the SAM layer is composed of low dielectric alkane chains 39 . A further increase in impedance, albeit a smaller change was observed in the subsequent modification of the IDE surface with APBA and anti-S. sclerotiorum antibodies. To illustrate the observed impedance change following different stages of the IDE surface modification, the magnitudes of the impedance recorded at 1000 Hz (in Fig. 2a) are plotted in Fig. 2b.
The impedance change observed upon conjugation of APBA and anti-S. sclerotiorum antibody to the covalently attached SAM as displayed in Fig. 2 indicates that the fabricated sensor has the sensitivity to detect the binding of antigens to the surface immobilized antibodies. In the affinity based biosensors, a major source of interfering noise comes from nonspecific binding of biomolecules present in the solution. Although, the nonspecific binding on the sensor surface can be greatly reduced by the formation of SAM layer on the IDE 40 , further treatment of the surface with reagents such as a blocking agent is required to minimize their effects. In our biosensor, the IDE after surface modifications and anti-S. sclerotiorum antibody immobilization was incubated with 2% BSA solution to minimize nonspecific binding prior to treatment with the target ascospore solution.
Since the selectivity and specificity of an impedimetric biosensor depend solely on the affinity of the probe antibody to the target antigen, and as the transducer generates an output signal to any particles, pollens, fungi or bacteria that attached/binds to the antibody, the chances of false positive signal exist unless the antibody has low or no affinity these adventitious materials. The anti-S. sclerotiorum antibody used in this study was produced and purified by Cedarlane Labs (Burlington, ON, Canada, https://www.cedarlanelabs.com/). The specificity against S. sclerotiorum was confirmed by ELISA method. Specificity test perform in our lab showed that the polyclonal antibody has high affinity for the target antigen, Sclerotinia sclerotiorum, as expected and encouragingly displayed little or no affinity for the fungus, Leptosphaeria maculans, which is responsible for blackleg, another devastating fungal disease of canola. This antibody was intended to be used as a probe for determining the biosensor perimeters for Sclerotinia stem rot disease in laboratory study. The specificity and the possible cross reactivity of anti-S. sclerotiorum antibody with fungi commonly found in crop fields have been investigated by a number of groups [41][42][43][44] . Jamaux and Spire 41,42 reported that anti-S. sclerotiorum polyclonal antibodies cross react with closely related species such as S. trifoliorum and S. minor, but do not cross-react with 30 other fungi which could possibly be found in different crop fields such as canola, wheat, sunflower, lettuce, soybean, etc. However, according to Bom and Boland 43 , the cross-reactivity among Sclerotinia spp. is not anticipated to be a significant source of error in the field because S. homoeocarpa, S. trifoliorum and S. minor are not typically found in the phyllosphere of canola due to their lack of spores, their host range, or both. The cross-reaction to Botrytis cinerea 41,42 may cause problem in the polyclonal anti-S. scleroriorum antibody application. To overcome the cross-reactivity problem two approaches could be adopted to improve the antibody specificity for field application viz to perform absorption of S. sclerotiorum antigen with polyclonal anti-B. cinerea antibody as described by Jamaux and Spire 41 or to develop monoclonal antibodies. Figure 3 shows the optical microscopy images of the ascospores selectively captured from ascospores in solution, via antigen-antibody affinity binding, by the immobilized antibodies on the surface of the IDE. It is important to note that the ascospores captured by the immobilized antibodies on the surface of the IDE are unaffected by repeated washing, indicating suitability of the boronic ester bond for antibody immobilization 18,21,24,25 . As the ellipsoidal shaped ascospore has size in the range, 4-6 µm × 9-14 µm 45 , a digital optical microscope was used in this work as a technique to verify the efficacy of the protocol used to capture ascospores by the immobilized antibodies.
The microscope images were also used for determining the number of ascospores captured on the IDE surface and to correlate their contributions to the impedance response. As shown in Fig. 3, the estimated number of ascospores in the representative images correspond to about 9700, 4000 and 2300 ascospores captured on the surfaces of 3 mm × 3 mm IDEs in the sensor chip. In this work, the numbers or concentrations of suspended ascospores in the solution were determined by using a hemocytometer. From the concentration of ascospores in the solution and the observed number ascospores captured on the surface of the IDE, we have determined that the A primary goal of the present work is to establish whether an impedance based non-Faradaic biosensor is suitable for sensitive detection of ascospores in solution and eventually to develop a viable technology for field applications suitable for remote onsite sensing and forecasting of Sclerotinia stem rot epidemic of canola. Figure 4 shows the plot of impedance change (ΔZ) versus ascospores concentration [ascospore] in the incubation solution.
The impedance change (ΔZ) at any given ascospore concentration was calculated from the impedance spectra of an IDE recorded before (Z Ab ) and after (Z Ab-Sp ) the capture of the ascospores by the immobilized anti-S. sclerotiorum antibodies on the surface of the functionalized IDE. Binding of the ascospores on the IDE surface led to a decrease in impedance magnitude, thereby yielding a negative value for the impedance change, ΔZ = Z Ab-Sp − Z Ab , calculated from the impedance magnitude at the applied sinusoidal AC frequency of 1000 Hz. As demonstrated by the experimental data plotted in Fig. 4, the impedance magnitude decreases with increase in the number of bound ascospores on the surface of the IDE.
The change in the magnitude of impedance in a capacitive based sensor due to affinity binding of target antigens to the surface immobilized antibodies can arise from the dielectric properties of the target antigens, the displacement of water molecules due the target binding, and the change in the thickness of the recognition layer [26][27][28][29][30][31][32][33][34] . These changes contribute to impedance change via the capacitance of the recognition layer (C REC ) in the biosensor.
A unique advantage of working with ascospores is their size 41 (4-6 µm × 9-14 µm) which allowed us to confirm the selective capture of ascospores on the sensor surface using optical microscope (Fig. 3). Further, the optical images were used to estimate the number ascospores captured on the IDE surface which in turn is related to the concentration of ascospores in the incubation solutions and these concentration values were plotted as shown in Fig. 4. Hence, these results allowed us to obtain a direct and unambiguous correlation between the number of selectively captured ascospores and their impedance response.
The experimental data in Fig. 4 can be fitted with a linear regression (R2 = 93%) to yield a slope of 6.5 ohms/ aM ascospores in the solution. The limit of detection (LOD) evaluated from the signal-to-noise ratio determined from the standard deviations of the negative controls was about 130 aM.
In the traditional convention for the expression of LODs for biosensors, the non-Faradaic biosensor developed in this work has a LOD of 7.8 × 10 4 ascospores/mL. The performance of the biosensor compares well with the LOD of 7.4 × 10 4 CFU/mL reported by Varshney and Li 46 in 2007 for the detection of E. coli O157:H7 by impedimetric biosensor using IDE as the transducer. However, in the past few years a significant progress has been achieved in lowering the LOD, and the state-of-the-art IDE impedimetric biosensor reported by the same group in 2015 has LOD of 100 CFU/mL 47 for the detection of E. coli with assay time as short as 1 hour. In spite of the general conception that IDE offers a better signal-to-noise ratio and sensitivity compared to the biosensors with traditional macro-electrode, it is interesting to note that LOD as low as 3 CFU/mL 48 for the detection of Salmonella Typhimurium has been reported for impedimetric biosensors using macro-electrodes as the transducers with assay time as short as 45 minutes. The impressive LODs achieved by these state-of-the-art IDE impedimetric biosensors are comparable to that of the LODs attained by advanced PCR methods for E. coli detection 49 . It is however important to point out that caution should be exercised when comparing LODs reported for E. coli detection because of the bacterium propensity to exponentially multiply to form a colony of huge number bacteria in the nutrient rich medium. In fact, using nutrient rich media, Settu et al. 50 has reported LOD of 7 cells/ mL for the detection of E. coli. In this respect it will be useful to note that the ascospores, the pathogen of canola, can germinate and grow to produce mycelium enabling them to infect canola plants but cannot multiply like E. coli to form colonies of ascospores. These differences can have significant impact on the biosensor sensitivity if a monoclonal antibody with epitope specific to the ascospores is used in the detection of Sclerotinia sclerotiorum. Although the LOD achieved in this work is significantly lower compared to the current methods such as qPCR used for monitoring airborne ascospores [12][13][14][15][16][17] , impedimetric biosensors have many advantages in terms of cost, miniaturization, automation, and onsite remote sensing capability. In addition, to the best of our knowledge this is the first report on an impedimetric biosensor for ascospores detection and thereby has huge potential for further improvements in sensitivity and LOD.
Even with the present level of performance, the impedimetric biosensor presented in this work could be used, and is being tested, for the forecasting of the outbreak of Sclerotinia stem rot, because airborne ascospores can be readily captured by high throughput ascospore traps. The threshold number of ascospore in the air which allows an 8-day advanced forecasting of the outbreak of Sclerotinia stem rot 12 is about 9 ascospores/m 3 . By pre-concentrating the ascospores with a spore trap, the impedimetric biosensor describe in this work can be used for detection and forecasting of the Sclerotinia stem rot outbreak and serves as an early warning system for the management and control Sclerotinia stem rot of canola.

Conclusions
The paper presents the development of a novel label-free capacitive biosensor for the detection and quantification of ascospores, a pathogen which causes canola stem rot, the most destructive disease for canola crop. The device was fabricated by a functionalization protocol designed to achieve oriented immobilization of the antibodies to attach covalently to the SAM modified surfaces of the gold electrodes of the IDE by using boronic acid chemistry. Oriented immobilization affords a high density of non-denatured probes available for binding with the target ascospores, and the high affinity of the antibodies for the ascospores provides the selectivity and specificity of the biosensor. The biosensor has been determined to have sensitivity and LOD for ascospores of 6.5 ohms/aM and 130 aM. The LOD of 130 aM or 7.8 × 10 4 ascospores/mL obtained for the developed biosensor is much higher compared to the LOD of 3 CFU/mL reported for state-of-the-art biosensors for the detection of food pathogens. This indicates that the biosensor for ascospores detection has a huge potential for further improvement and also highlighted the need for the development of a monoclonal antibody with a high affinity for a specific epitope of the ascospores and possessing no cross reactivity with all other fungi or pollens present in the field where canola is grown. Based on the performance of the state-of-the-art biosensors, it is not unreasonable to expect the development of a biosensor optimized to a level with the capability to detect ascospores with LOD of 10 ascospores/ mL and hence to provide a biosensor with the ability to forecast the outbreak of the Sclerotinia stem rot epidemic of canola.

Materials and Methods
Material and reagents. 1-Mercapto-11-undecanoic acid 97% (11-MUA), 1-Mercapto-6-hexanoic acid 90% Polyclonal anti-S. sclerotiorum antibody was produced by Cedarlane lab following a standard procedure from rabbits using S. sclerotiorum as the antigen. Ascospores of S. sclerotiorum were produced using a standard method (InnoTech Alberta accession #184) by planting sclerotia, generated from sliced carrot roots, into a wet sand and incubating at 10 °C until the sclerotia germinates to form apothecia. The ascospores released from the apothecia were harvested by trapping onto a filter paper disc by applying vacuum.

Gold IDE sensor chip and Polydimethylsiloxane (PDMS) mask. A custom designed IDE sensor chip,
with digit parameters optimized for nanoparticle-enhanced impedimetric sensors 51 , was fabricated on a silicon wafer with 500 nm thermal oxide following a standard photolithography process flow involving sputter deposition of chromium (10 nm) and gold (100 nm), photoresist and photomask pattern transfer followed by development, reactive ion etching and lift-off. Each of the sensor chips has eight 3 mm × 3 mm square IDEs with digit length, width, thickness, and gap of 3 mm, 3 µm, 110 nm, and 3 µm respectively. Images of a typical sensor chips is displayed in Fig. 5.
To facilitate functionalization of the IDE surface without affecting other areas of the chip, a PDMS mask, as shown in Fig. 5b, with eight units of 3 mm × 3 mm square wells, designed to fit the IDEs on the sensor chip was custom designed and fabricated in the lab. The PDMS mask allows independent and localized modification of each IDE on a sensor chip with any desired modifying solutions.
Protocols for Surface functionalization. The chips used for surface functionalization were cleaned by sonicating for about 5 minutes each in acetone, isopropanol, Millipore water (MPW) and then drying with a stream of nitrogen. The chips were then exposed to Argon plasma (1 Torr Ar atmosphere, 18 W high RF) for 5 minutes to ablate any adsorbed organic materials on the surface. The freshly cleaned IDEs of the chip were functionalized in a sequence of successive reaction steps by submerging them in different solutions using the PDMS mask as presented in Fig. 5. After each functionalization reaction step, the IDEs were washed with the solvents used to prepare the solutions including ethanol, 10 mM PBS at pH 7.4 and/or MPW to remove any chemicals not covalently bound to the surface.
The affinity of thiol with gold was utilized to form an insulating self-assemble monolayer (SAM) with distal carboxylic acid group on the surface of the IDEs. The reaction was carried out by submerging the IDE overnight Instruments. Electrochemical Impedance Spectroscopy (EIS) measurements were performed with a potentiostat/galvanostat SP-200 controlled by EC lab software from BioLogic Scence Instruments Inc (Knoxville, Tennessee). A custom built electrical contact pad and connector, as shown in Fig. 5b, was used to make electrical connections between SP-200 and the IDEs on the sensor chips. A PDMS mask with eight wells was used to submerse the IDEs of the sensor chip with 50 µL of 10 µM PBS at pH 7.4 for impedance spectra measurements. Impedance spectra were measured by applying 10 mV sinusoidal excitation perturbation at 0 V DC in the frequency range of 10 Hz to 1 MHz.
A digital optical microscope, VHX-700F from KEYENCE Canada Inc. (Mississauga, Ontario), was used for imaging and estimating the number of ascospores captured on the surface of the IDEs. A Hemocytometer and Motic AE 31 An inverted Biological Microscope (Carlsbad, California) was used to determine the number and concentration of ascospores in the re-suspended solutions.
Data availability. The data files including raw data used to support the findings in this study can be obtained from the corresponding author upon reasonable request. Figure 5. Sensor chip, PDMS mask, electrical contact pad, and electrical connector. The sensor chip has eight units of 3 mm × 3 mm square IDEs (a) and the matching PDMS mask has eight wells (b) to fit the IDEs and individually functionalize them. Each gold electrode finger or digit in the IDE has length, width, thickness, and gap of 3 mm, 3 µm, 110 nm, and 3 µm respectively.