---
title: "Photonic crystal surface mode imaging for multiplexed and high-throughput label-free biosensing"
authors: ["Elseviermyfootnote", "Elsevier Inc", "Valery Konopskycor1", "Tatiana Mitko", "Konstantin Aldarov", "Elena Alieva", "Dmitry Basmanov", "Aleksandr Moskalets", "Ainur Matveeva", "Olga Morozova", "Dmitry Klinov"]
affiliation: "Institute of Spectroscopy, Russian Academy of Sciences, Troitsk, Moscow region, Russia."
journal: "Biosensors and Bioelectronics"
year: 2020
volume: ""
issue: ""
article_number: ""
pages: ""
doi: ""
type: journal-article
site_group: ""
url_abstract: ""
url_pdf: "https://valery.konopsky.com/kvnlocal/Konopsky2BB_rev.pdf"
language: en
source_tex: "Z:\\ValeryData\\Valery_New\\my_articles\\BiosensorsBioelectronics2020\\BiosensorsBioelectronics\\manuscript\\Konopsky2BB_2020_2.tex"
source_pdf: "Z:\\ValeryData\\Valery_New\\my_articles\\BiosensorsBioelectronics2020\\BiosensorsBioelectronics\\published\\Konopsky2BB_rev.pdf"
---
## Abstract

A photonic crystal surface mode imaging (PC\,SM{\it i}) technique is implemented for the simultaneous detection of antibody binding with specific antigens in arrays containing 96- and 384-spots. Like the surface plasmon resonance imaging (SPR{\it i}) technique, the presented approach is label-free and permits interrogating an analyte by hundreds of different ligands immobilized in small spots. The adsorption kinetics is recorded with a sub-picogram resolution at every spot simultaneously. Possible implementations of this technique for multiplexed and high-throughput biosensing are discussed.

label-free optical biosensors ,surface wave imaging ,photonic crystal surface modes ,SPRi ,PCSMi

# Introduction

A comparative assessment of affinities and binding kinetics of biomolecular complexes is important for drug discovery and pharmaceutical development, as well as for basic biology and proteomics. Optical biosensors can provide such an assessment in real time without fluorescent labels, which can introduce unwanted detrimental effects, for example, interference with the fundamental interaction [4].

The most popular type of label-free optical biosensors is the surface plasmon resonance (SPR) sensor, which was developed more than a quarter century ago [13, 12]. Currently, most commercial label-free optical biosensors are based on the SPR effect [21]. An imaging modification of the SPR biosensors (SPR*i*) was also demonstrated in the late 1980s by two groups: Yeatman and Ash [23] and Rothenhäusler and Knoll [16, 17]. These SPR*i* biosensors have two-dimensional (2D) spatial resolution and have recently attracted much attention [18, 20, 22, 1]. The 2D spatial resolution of the imaging version of a biosensor allows depositing a large number of capture antibodies on a sensing surface and interrogating an analyte by them simultaneously. Commercial SPR*i* devices are also available, for example, from Biacore [2] [Flexchip [15]], GWC Technologies [5], HORIBA Scientific [6], and is used in such fields as high-throughput biosensing and chemical sensor arrays.

Besides this popular SPR technique, which is based on the excitation of surface plasmon (SP) waves on a metal surface, a new label-free biosensing technique based on the excitation of photonic crystal surface modes (PC SMs) on the surface of a dielectric multilayer has recently attracted much attention [19, 8, 9, 11]. The propagation length of PC SMs is one to two orders of magnitude longer than the propagation length of SPs (due to the absence of dissipation of optical energy in the metal) and the sensitivity of the PC SM biosensing technique is therefore potentially higher.

Recently, an imaging modification of the PC SM biosensor (PC SM*i*) with a dynamic range and sensitivity comparable to or better than those in the SPR*i* biosensor, was introduced [10]. In this approach, the spectral shift of the PC SM resonance is recorded by a color camera with 2D spatial resolution. This permits simultaneously interrogating an analyte by a large number of capture antibodies, which are spotted as a microarray on the surface of a PC chip.

In the present article, we demonstrate the simultaneous detection of biochemical reactions in 96 and 384 spots using the PC SM*i* biosensor.

# Materials and methods

## Chemicals and reagents

(3-Aminopropyl) triethoxysilane (APTES), bovine serum albumin (BSA), glutaraldehyde (GA), hIgG and rabbit polyclonal antibodies against hIgG (anti-hIgG) were purchased from “Sigma-Aldrich” (USA). Hepatitis B virus surface antigen of subtype ayw (HBsAg-ayw) was purchased from “Bialexa” (Russia), mouse monoclonal antibody HV-42 against determinant “a” of HBsAg (anti-HBsAg HV-42) was purchased from “BioSan” (Russia).

A 5% APTES solution and 0,1% GA solution were prepared with double-distilled water. BSA was dissolved in PBS to obtain a solution with a concentration 0.1 mg/mL. Antigens were dissolved in 2% DMSO water solution to obtain solutions with concentrations 0.05 mg/mL for hIgG and 0.15 mg/mL for HBsAg-ayw. Monoclonal and polyclonal antibodies were dissolved in PBS for binding with antigen.

## Photonic crystal structure

Te PC chips used in our experiments have the following structure: *substrate* $/H(LH)^{3}L'/$ *water*, where $H$ is a $\mathrm{TiO_2}$ layer (thickness $d_2=63.1$ nm), $L$ is a $\mathrm{SiO_2}$ layer (thickness $d_1=240.2$ nm), and $L'$ is a final $\mathrm{SiO_2}$ layer (thickness $d_3=341.0$ nm). The $\mathrm{TiO_2}/\mathrm{SiO_2}$ 8-layer structure, with $\mathrm{TiO_2}$ as the first layer and $\mathrm{SiO_2}$ as the last layer, was coated by a SYRUSpro 710 optical vacuum coater (Buhler Leybold Optics, Alzenau, Germany) via electron-beam evaporation and plasma ion-assisted deposition. The prism and the glass plate substrate were BK-7 glass. At $\lambda=500$ nm, the RIs of the substrate, $\mathrm{SiO_2}$, $\mathrm{TiO_2}$, and water are respectively $n_0=1.521$, $n_1=n_3=1.475$, $n_2=2.433$ and $n_e=1.338$. The presented multilayer PC structure supports a $p$ -$\!\!$ polarized PC surface mode at $\lambda=500$ nm with an effective refractive index (RI) $\rho=n_0\sin(\theta_0)=1.3812$, corresponding to an in-prism excitation angle $\theta_0=65.2^0$.

## Sample preparation

Before the experiment, the PC chip was thoroughly rinsed with double-distilled water and ethanol was then treated in the plasma cleaner Zepto W6 (13,56 MHz/100 W, Diener Electronic, Germany) for 10 minutes at an air pressure 600 to 800 mbar. After that the PC chip was immersed in APTES solution for 30 minutes, rinsed with double-distilled water, and baked for 30 minutes at 120$^{\circ}$C. The surface of the PC was then covered by GA solution and rested for 30 minutes. Further, the PC chip was rinsed with double-distilled water.

The PC chip with a modified surface was placed in the spotter iTwo-300P (M2-Automation, Germany). This device allows applying the antigens to the surface according to a set pattern. To prepare a microarray containing 384- (96-) spots, 200 (300) drops (60 pL each) were applied to each spot on the surface. The reagent concentration in the drops was 0.05 mg/mL for hIgG, and 0.15 mg/mL for HBsAg (ayw). Spots were deposited at a 20$^{\circ}$C, and humidity was not controlled. The resulting spot diameter was about 430 $\mu$m. The sample was then mounted in a biosensor flow cell with a height of about 12 $\mu$m. To block unbound GA on the surface of the PC chip, BSA was flowed over the flow cell for several minutes, and the system was then thoroughly rinsed with PBS. After that antibodies against hIgG and HBsAg were injected into the system sequentially.

![Schematic of the PC SMi biosensor. Typical spectra of light as it passes from the LED to the color camera are shown.](media/BiosensorsBioelectronics2020/Fig1.eps)

*Schematic of the PC SMi biosensor. Typical spectra of light as it passes from the LED to the color camera are shown.*

# Results and discussion

## Operation of PC SM imaging biosensor

The basic diagram in Figure 1 shows the principle of operation of the label-free PC SM*i* biosensor. A wide parallel light beam, with polarization $+45^0$ to the plane of incidence, emitted by a fiber-coupled LED, with a peak wavelength near $\lambda=500$ nm and a bandwidth (FWHM) of 35 nm is used to excite the PC SM. The PC SM is excited on an external surface of a one-dimensional (1D) PC chip through a coupling prism (Kretschmann-like configuration). After total internal reflection (TIR) from the PC chip, the light beam passes through the second polarizer at $-45^0$ to the plane of incidence, and is then recorded by a color camera. The PC SM excitation causes a phase shift of the surface wave that re-radiates back to the prism. Consequently, the polarization of the re-radiated surface wave rotates, and only light at the excitation wavelength of the PC SM passes through the crossed polarizers and enters the color camera.

The mean wavelength of the light that has passed through is determined by balancing the intensities of the blue and green pixels at each point in the color camera. The structure of the 1D PC was designed such that the excitation wavelength of the PC SM is located between the maxima of the blue and green pixels. These spectral positions are illustrated schematically in Fig. 2. The figure shows that an increase or decrease in the adsorption layer thickness $d_{\mathrm a}$ causes a shift in the PC SM peak to respectively longer or shorter wavelengths, thus increasing or decreasing the green component and simultaneously decreasing or increasing the blue component in the color pixel response.

![PC SM peak (red line) and responsivities of the green and blue pixels of D1312C camera, provided by the manufacturer (Photonfocus, Lachen, Switzerland).](media/BiosensorsBioelectronics2020/Fig2.eps)

*PC SM peak (red line) and responsivities of the green and blue pixels of D1312C camera, provided by the manufacturer (Photonfocus, Lachen, Switzerland).*

Data acquired by the color camera was processed and presented using custom software. The shift of the PC SM resonance wavelength was determined from the next normalized differential value:
``` math
\begin{equation}
   z(x,y)=\frac{B(x,y)-G(x,y)}{B(x,y)+G(x,y)}\, ,
   
\end{equation}
```
where $B(x,y)$ and $G(x,y)$ are the intensities of the blue and green components in each color pixel $(x,y)$ of the camera.

## Simultaneous registration of the kinetics of biochemical reactions in a 384-spot microarray (natural colors)

To demonstrate the multiplexing potential of the PC SM*i* technique, we recorded the reaction kinetics simultaneously at 384 spots on the PC biochip surface. For this test, we used two different binding reactions: 187 spots of human immunoglobulin G (hIgG) and 188 spots of the hepatitis B virus surface antigen (HBsAg) were deposited onto the surface in chess-board order using a spotter (see section 2 for details).

![Images (in natural colors) of the 384-spots microarray before (79 sec) and after (1637 sec) injections of antibody solutions. Seven spots (#38, 86, 134, 182, 230, 278 and 326) are empty and used as a reference. Kinetic curves for two neighbouring spots (#48 and #64) are shown in the bottom.](media/BiosensorsBioelectronics2020/Fig3.eps)

*Images (in natural colors) of the 384-spots microarray before (79 sec) and after (1637 sec) injections of antibody solutions. Seven spots (#38, 86, 134, 182, 230, 278 and 326) are empty and used as a reference. Kinetic curves for two neighbouring spots (#48 and #64) are shown in the bottom.*

Seven spots (38, 86, 134, 182, 230, 278 and 326) remained empty as a reference. The results are presented in Fig. 3. The vertical and horizontal shift between the spots is 500 $\mu$m. The round spots in the figure appear as elliptical because the whole image is squeezed in one dimension in a ratio of about 2:1. This image distortion is the result of reflection of the beam inside the prism at an angle of $\theta_0=65.2^0$.

Images from the camera in Fig. 3 are shown in their natural color. The biosensor detects a slight color change at each point of the camera using normalized difference (1) and recalculates it as a $\Delta\lambda$ shift, as described in [10]. Typical kinetic curves for two adjacent spots (#48 and \#64) are shown in the bottom of Fig. 3. A solution with the rabbit polyclonal antibodies against hIgG (diluted to $75~\mu$g/ml) was injected at 108 sec. At 739 seconds, a pure PBS solution was added for removing anti-hIgG and clearing, and then a solution of mouse monoclonal antibodies HV-42 against the determinant “a" of human HBsAg (diluted to $52~\mu$g/ml) was injected at 968 sec. A final injection of PBS was added at 1637 sec.

From these kinetic curves one can see that a single spot reacts with the corresponding antibody only when it is introduced in the analyte solution. The mean value from blank reference spots has been subtracted from each kinetic curve to reduce the effects of non-specific adsorption and refractive index (RI) changes from injected solutions.

It can be also seen from Fig. 3 that, despite a slight color change that is difficult to notice in the images with the naked eye, the use of equation (1) allows registering a subnanometric change in the wavelength with a good signal-to-noise ratio. To increase the visible contrast of the images, we can use a pseudo-color (based on  (1)) instead of the natural color. This does not change the kinetic curves, whose registration is based on equation (1) in any case, but it does make changes in the images more noticeable to the naked eye. An example of this is given in the next subsection.

## Simultaneous registration of the kinetics of biochemical reactions in a 96-spot microarray (pseudo-color)

An array containing 96-spots of immunoassay deposited on the PC chip, is used in this demonstration experiment. It contains three reference spots (#19, 43, and 67) that are used for subtraction from the signal to reduce the effects of non-specific adsorption and bulk RI changes, 46 spots of HBsAg and 47 spots of hIgG (see section 2 for details). The horizontal shift between the spots during deposition is 900 $\mu$m, and the vertical shift is 1000 $\mu$m. The results are shown in Fig. 4 and in the video presented in Supplementary data.

![Images (in pseudo-color) of the 96-spots microarray before (17 sec) and after (1532 sec) injections of antibody solutions. Three spots (#19, 43, and 67) are empty and used as a reference. Kinetic curves for two neighbouring spots (#56 and #64) are shown in the bottom. A complete set of images during adsorption is presented as a video in Supplementary data.](media/BiosensorsBioelectronics2020/Fig4.eps)

*Images (in pseudo-color) of the 96-spots microarray before (17 sec) and after (1532 sec) injections of antibody solutions. Three spots (#19, 43, and 67) are empty and used as a reference. Kinetic curves for two neighbouring spots (#56 and #64) are shown in the bottom. A complete set of images during adsorption is presented as a video in Supplementary data.*

It can again be seen from the kinetic curves in Fig. 4 that one spot with a particular antigen responds only to the specific antibody when it is introduced in the analyte solution. In this experiment, the solution with anti-hIgG (diluted to $75~\mu$g/ml) was injected at 55 sec, a pure PBS solution (for removing anti-hIgG and clearing) was added at 412 sec, and the solution of anti-HBsAg-ayw (diluted to $52~\mu$g/ml) was injected at 570 sec. A final injection of PBS was added at 1533 sec.

It can be see that using the pseudo-color instead of natural colors permits noticing even small changes in the thickness of the adsorbate with the naked eye. If only one or several spots would react with an analyte, their position would be seen not only from numeration of the kinetics curves, but also from the image directly.

The limit of detection (LoD) in a single spot of the biosensor is determined by the baseline noise, which is $\delta\lambda=7\cdot10^{-4}$ nm in the presented kinetic curves. This corresponds to the adlayer thickness noise $\delta d_a =2.5\times10^{-3}\,\mathrm{nm}=2.5\,\mathrm{pm}$, calculated using the sensitivity of this biosensor to changes in adlayer thickness, which is $\Delta\lambda/\Delta d_a=0.284$ [10]. Such thickness noise corresponds to 2.5 pg/mm$^2$ in terms of surface mass density. For the spot size $430\times430$ $\mu$m$^2$ this gives 460 fg for a single spot. Therefore, about 1.4 pg of analyte should be immobilized in one $430\times430$ $\mu$m$^2$ spot to be detected with a signal to noise ratio of 3.

## Comparison with other methods

Drug design, preclinical trials and further implementation are required biomedical research of numerous interactions of chemical compounds with their potential molecular targets, supramolecular structures, viruses and cells. Currently available, widely used methods include enzyme-linked immunosorbent assay (ELISA), immunofluorescent analysis with magnetic microspheres (xMAP) [14], reverse transcription with real time PCR, mass parallel sequencing and various biological tests. The sensitivity limits of ELISA and xMAP are near 1 pg/ml, whereas PCR permits detecting a single molecule in a reaction mixture. But, as already mentioned, the labeling of biopolymers can cause steric hindrances for further specific interactions with other potential ligands or targets.

On the other hand, label-free methods (SPR*i* and PC SM*i*) not only detect the fact of interaction but also provide information about the kinetics of all reactions in hundreds of spots simultaneously. Accordingly, the evident advantages of the PC biosensor of this new (image) generation include real time multiplex label-free analysis of antibodies binding with specific antigens. To our knowledge, none of the currently available conventional methods permits combining all these features. Nevertheless, the relatively low sensitivity limit of the PC SM*i* technique (near 0.2 $\mu$g/ml of proteins), which is significantly less than the sensitivity limits of ELISA and xMAP immunofluorescent analysis (approximately 1 pg/ml), must be noted.

# Conclusions

We have demonstrated that the PC SM*i* technique can interrogate an analyte simultaneously by hundreds of captured antigens or antibodies spotted on 96- and 384-spot microarrays. Like SPR*i* biosensors, the imaging version of the PC SM biosensor can be exploited in a wide range of applications from the "electronic nose" for monitoring volatile organic compounds in a gas environment [3], to efficient selection of tight-binding target for the discovery of aptamers [7], to mention just a few recent implementations. Compared with SPR*i* biosensors, the sensitivity of the presented PC SM*i* biosensor is potentially higher because metal damping in the dielectric multilayers is absent.

# Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

# Acknowledgements

This research was supported by the Russian Foundation for Basic Research (project No 18-32-00797). The part of the work related to experiments on the photonic crystal surface modification technique was funded by the Russian Science Foundation (grant No 17-75-30064). We thank the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency for help with microscopic and fluorescence analysis of spotted microarrays.

## Appendix A: Supplementary data

A complete set of images during adsorption on 96-spot microarray is presented as a video file in Supplementary data.

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