LIPID NANOPILLAR ARRAY-BASED IMMUNOASSAY

Abstract

Provided are: an array, a kit, and/or a device which are for analyzing, identifying, detecting, and/or visualizing target particles, and/or determining the presence or absence of the target particles, and use a substrate having an uneven surface coated with a lipid bilayer; and a method using same.

Claims

1. An array for detecting a target particle, comprising: a substrate with uneven surface, a lipid bilayer coating the uneven surface, and plural capturing substances binding to the target particle, wherein the capturing substances are localized in the lipid bilayer with fluidity, and labeled with a signaling material.

2. The array for detecting a target particle of claim 1, wherein the uneven surface comprises a plurality of pillars, grooves, or a combination thereof.

3. The array for detecting a target particle of claim 2, wherein diameter, height, or interval of the pillar or groove is at least 1.2 times the average diameter of the target particle.

4. The array for detecting a target particle of any one of claim 1, wherein the substrate is a solid substrate having hydrophilic surface or modified to have hydrophilic surface.

5. The array for detecting a target particle of claim 1, wherein the target particle is at least one selected from the group consisting of viruses, cells, proteins, and nucleic acid molecules.

6. The array for detecting a target particle of claim 5, wherein the target particle is a virus.

7. The array for detecting a target particle of claim 6, wherein the target particle is a corona virus, an influenza virus, or a combination thereof.

8. A kit for detecting a target particle, comprising two or more of the arrays for detecting a target particle of claim 1.

9. The kit for detecting a target particle of claim 8, wherein the arrays are for detecting the same target particle.

10. The kit for detecting a target particle of claim 8, wherein the arrays are for detecting different target particles from each other.

11. The kit for detecting a target particle of claim 8, wherein the target particle is a virus.

12. The kit for detecting a target particle of claim 11, wherein the target particle is a coronavirus, an influenza virus, or a combination thereof.

13. A method of detecting a target particle, comprising: contacting a sample with the array for detecting target particle of claim 1 or a kit for detecting target particles comprising a plurality of the arrays; and measuring a signal generated from the array or the kit.

14. The method of claim wherein the sample is at least one selected from the group consisting of cells, blood, lymph, saliva, sputum, snivel, urine, and feces, obtained from a mammal.

15. The method of claim 13, wherein the target particle is a virus.

16. The method of claim 15, wherein the target particle is a coronavirus, an influenza virus, or a combination thereof.

17. (canceled)

18. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1a is a schematic view showing a process of a nanopillar-supported lipid bilayer immunosorbent assay (LNAIA) according to an embodiment of the present description and characteristics of the nanopillar-supported lipid bilayer substrate.

[0062] In FIG. 1B, h) schematically shows an experimental procedure of transmission electron microscopy (120 kV, Talos L120C, FEI, Hillsboro, United States) of the lipid bilayer formed on the nanopillar-patterned substrate, i) shows a TEM image of the micro-sectioned nanopillars, j) shows a SEM image of a tilted nanopillar substrate, k) shows SEM image of the pillar-to-flat boundary area, 1) shows a fluorescence microscopic image of 1% Texas Red-modified SLB formed on the pillar-to-flat boundary area, m) shows a fluorescence intensity line profile on the dotted line shown in FIG. 1a (e), and n) shows a fluorescence recovery after photobleaching (FRAP) results of the nanopillar patterned substrate and a flat substrate (diffusion coefficients of flat SLB and nanopillar SLB are ˜0.34 μm2/s and ˜0.35 μm2/s, respectively. The colored areas in the plots represent mean standard errors. The scale bars in i-j, and k-n are 200 nm and 5 μm, respectively).

[0063] FIG. 2 shows virus capture and mobility results on nanopillar and flat SLB substrate, wherein a) illustrating a trapped virus on the nanopillar SLB after assay, b) schematically shows a freely moving virus on a flat SLB after assay, c) is an epifluorescence microscopy images on lipid nanopillar array (LNA) before and after assay, d) is an epifluorescence microscopy images on flat SLB before and after assay, e) is a SEM image of substrate after assay, wherein the SEM image was acquired after freeze-drying, f) shows LNAIA results on each SLB with antibody (Ab-O) and without antibody (Ab-X) (the p-value between Ab-O and AB-X on the LNA was 0.0286. *p<0.05 (one-tailed Mann-Whitney U test)), g) shows trajectory of fluorescence spots on the nanopillar SLB via an epifluorescence microscope, h) shows trajectory of fluorescence spots on flat SLB via TIRF microscope, i) is a graph showing mean square diameter of fluorescence spots on each SLB after assay, and j) shows diffusion coefficient.

[0064] FIGS. 3a-c show LNAIA detection results for H1N1 virus as a target particle, wherein, 3a schematically shows an elimination of false positive results by weak fluorescence signal-based paucivalent nonspecific bindings, 3b is a graph showing LNAIA and ELISA results for H1N1 target and non-target viruses (1.5*10.sup.5 particles/chamber for LNAIA and 9.1*10.sup.5 particles/chamber for ELISA) (in LNAIA, the p-value for H1N1 compared to H3N2 and AdnV was 0.028. *p<0.05 (one-tailed Mann-Whitney U test)), and 3c is a graph results of quantitative analysis of H1N1 virus detection in human serum using LNAIA and ELISA (after virus loading, ELISA required roughly 6 hours while LNAIA required 25 minutes, including data analysis. Negative control was conducted with 1.5*10.sup.5 particles/chamber and 9.1*10.sup.5 particles/chamber of adeno virus for LNAIA and ELISA respectively, and the plotted data represent mean±standard deviation).

[0065] FIG. 4 shows a schematic diagram of a virus detection process using LNAIA (upper part), and an SEM image of the nanopillar substrate corresponding to the upper schematic diagram (lower part).

[0066] FIG. 5 shows comparison between the procedures of LNAIA and ELISA.

[0067] FIG. 6 shows a stepwise depiction of a LNAIA setup according to an embodiment of the present description.

[0068] FIG. 7 is a large-scale SEM image of a nanopillar pattern (Scale bar is 1 μm).

[0069] FIG. 8 is a tilted SEM image of a nanopillar substrate (Scale bar is 200 nm).

[0070] FIG. 9 is a TEM images of sectioned LNA (Scale bars are 1 μm).

MODE FOR INVENTION

[0071] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are only illustrative of the present invention, and the scope of the present invention should not be limited thereto.

Example 1. Preparation of LNAIA Kit

[0072] 1.1. Preparation of Small Unilamellar Vesicles (SUVs)

[0073] In a 50 mL round-bottomed flask, 97.45 mol % DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine), 0.05 mol % biotinylated DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), and 2.5 mol % PEG-DOPE (Molecular Probes, USA) were mixed in chloroform. The prepared lipid solution was evaporated with a rotary evaporator. For the FRAP experiment (Fluorescence recovery after photo bleaching) and fluorescence imaging of SLB (Supported lipid bilayer) in the pillar-to-flat boundary area, 1 mol % FITC-DHPE (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) and 1 mol % TexasRed-DHPE were included in the round-bottomed flask, respectively. Using a stream of N2 gas, the lipid film was completely dried. After being thoroughly dried, the solution was resuspended in DI water (Nanopure water with a minimum resistance >18 MΩcm.sup.−1), transferred to a cryo-tube, and subjected to three freeze-thaw cycles. The final lipid concentration was 4 mg/mL. The lipid solution was extruded 21 times through a polycarbonate (PC) membrane (Whatman, Fisher Scientific) with a pore diameter of 100 nm at 25° C. Liposomes of approximately 100 nm were obtained and kept at 4° C. until use.

[0074] 1.2. Antibody Biotinylation.

[0075] 0.2 mg/mL H1N1 antibody (Influenza A H1N1 (A/Puerto Rico/8/1934) Hemagglutinin, Sinobiological) in 150 mM PBS (50 μL) and 20 μM NHS-biotin (N-hydrocysuccinimidobiotin Thermo Scientific, USA) in DMSO (5 μL) were mixed and incubated for 2 hours at room temperature. Non-reacted biotin was removed with desalting columns (Zeba Spin desalting columns 7K MWCO, Thermo Scientific, Rockford, Ill., Rockford). The prepared reacting product described above was loaded in washed desalting columns and 150 mM PBS was added as a stacker. The column was centrifuged at 1500 g for 2 minutes in eppendorf tube, and the biotin-modified antibody in the solution collected bottom of the tube was bound to streptavidin (STV) on the lipid bilayer.

[0076] 1.3. Nanopillar Pattern Fabrication Using Spin-On-Glass and Nanoimprint Lithography

[0077] A 4-inch glass wafer was cleaned with plasma asher for 10 min. The glass substrate was spin-coated with IC3-200 spin-on-glass (SOG, Futurrex Inc. USA) at 3000 rpm for 30 sec. The hole-patterned polydimethylsiloxane (PDMS; SYLGARD 184, Dow Corning, USA) stamp was 200 nm in diameter, 300 nm in depth, and 400 nm in pitch, and was pressed onto the SOG-coated glass wafer with 2000 kgf cm.sup.−2 at 150° C. for 10 minutes using ANT-6H02 UV/thermal nanoimprint lithography (KIMM, Korea). Afterwards, the hole stamp was detached from the glass wafer, and a substrate with nanopillars formed on its surface (hereinafter, called as ‘nanopillar substrate, nanopillar patterned substrate, or nanopillar array’) was obtained (FIG. 1a (a)).

[0078] 1.4. Fabrication of LNAIA Kit

[0079] To fabricate a virus assay kit, silica nanopillar-patterned substrate was given hydrophilicity by treating the silica nanopillar-patterned substrate prepared in Example 1.3 with plasma asher after washing with ethanol and acetone for 10 times respectively. On the substrate, a sticker chamber (8 μL) was attached and small unilamellar vesicles (SUVs) prepared in Example 1.1 were mixed with 150 mM phosphate buffered saline (PBS) at the ratio of 1:1 (v/v) and introduced to the chamber for 30 minutes. After marking a cross line on the bottom of each chamber with a disposable needle and removing excess SUV with 150 mM PBS, 8 μL of 20 μM bovine albumin serum (BSA, Sigma Aldrich) in 150 mM PBS was introduced to inactivate the non-covered area on the nanopillar-patterned substrate. After removing excess BSA with 3 washes with 150 mM PBS, 8 μL of 20 nM Cy3-modified streptavidin (Cy3-STV, Molecular Probes, USA) in 150 mM PBS was introduced for 30 minutes, followed by another wash with 150 mM PBS. Subsequently, biotinylated antibody (Example 1.2) with an O.D. of 0.02 at 280 nm was introduced for 30 minutes. The chamber was then washed three times with 150 mM PBS prior to the assay.

Example 2. LNAIA

[0080] A LNAIA was performed using the LNAIA kit (target virus: H1N1) prepared in Example 1.4. A step wise experimental procedure and LNAIA chamber design is illustratively described in FIGS. 5 and 6. Epifluorescence images of 4 quadrants from each chamber were acquired before and 20 minutes after introducing 8 μL of 1.8*10.sup.4 to 1.8*10.sup.9 viral particles/mL virus (Influenza A/Puerto Rico/8/1934 H1N1 for positive target, and Adenovirus type 5 for negative target) sample with 1% (v/v) human serum (from human male AB plasma, USA origin, sterile-filtered, Sigma-Aldrich) in PBS to the LNAIA chamber under exposure to a 488 nm laser and 60× lens via TE-2000 (Nikon, Tokyo, Japan). Three images with an 80×80 μm.sup.2 (512×512 pixel.sup.2) field of view and 100 ms exposure time were stacked. The fluorescence images were analyzed to count ΔN (the increased number of fluorescent spots in the four quadrants) using the MOSAIC plugin for Image J software. The four ΔNq (the increased number of fluorescence spots in each quadrant) from a single well were summated as ΔN.

Reference Example 1. ELISA (Comparative Example)

[0081] 50 □L of 1.8*10.sup.4 to 1.8*10.sup.9 viral particles/mL virus (Influenza A/Puerto Rico/8/1934 H1N1 for positive target, and Adenovirus type 5 for negative target) samples with 1% human serum in coating buffer (0.2 M Na.sub.2CO.sub.3/NaHCO.sub.3, pH 9.6) were added to individual wells and incubated for 2 hours at room temperature. The wells were washed with 200 □L of PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 0.1 w/v % Tween 20) 3 times and patted dry on a hand towel. The remaining surface of each well was blocked with 200 □L of 1% BSA in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4) for 2 hours at room temperature. Excess BSA was removed with two washes of 200 □L of PBST. 2 □g/mL of primary antibody in PBS with 1% BSA was introduced to each well in a total volume of 100 □L for 2 hours at room temperature. The excess antibody was removed with 4 washes with 200 □L of PBST. 100 □L of secondary antibody with 1% BSA in PBS was added and incubated for 2 hours at room temperature. The wells were washed 4 times with 200 □L PBST and 50 □L of 3,3′,5,5′-tetramethylbenzidine (TMB) was added. After 15 minutes, each well was treated with 50 □L of 2 M of H2504 and analyzed by a microplate reader (Multiple Plate Reader, Victor 3, Perkin Elmer, USA).

Reference Example 2. Transmission Electron Microscopy (TEM) Assay

[0082] For imaging SLB structure on a nanopillar pattern, the nanopillar labyrinth SLB was fixed in 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (SCB) (pH 7.3) at room temperature for 30 minutes, followed by fixation overnight. The following steps were carried out at 4° C. until graded ethanol series. The sample was washed in 0.1 M SCB with 0.2 M sucrose 3 times and post-fixed with reduced osmium (1% osmium tetroxide with 0.8% potassium ferricyanide in 0.1 M SCB). After washing with distilled water 3 times, en-bloc staining was carried out with 2% uranyl acetate. After washing the sample with distilled water 3 times, it was dehydrated with 30%, 50%, 70%, 80%, and 100% ethanol in series. Next, the ethanol was exchanged for acetonitrile. Sample infiltration was carried out with a 1:1 mixture of acetonitrile and Spurrs' resin for 2 hours, a 1:2 mixture of acetonitrile and resin for 2 hours, and pure resin overnight at room temperature. The resin was exchanged with fresh resin and baked at 70° C. overnight. The bottom silica nanopillar pattern was etched away with 49% hydrofluoric acid (Sigma Aldrich) in a fume hood, thoroughly washed with distilled water, and dried at 70° C. for 30 minutes. The sample was re-embedded with pure resin and baked at 70° C. overnight. Subsequently, the resin block was sectioned by ultramicrotome (EM UC7, Leica, Wetzlar, Germany) into 70 nm-thick sections, then collected onto a TEM copper grid (Veco Center Reference Square Grid, 200-mesh, Cu, Tedpella, USA) and post-stained with 2% uranyl acetate. The prepared grids were imaged with 120 kV transmission electron microscope (Talos L120C, FEI, Hillsboro, United States).

Reference Example 3. Scanning Electron Microscope (SEM) Assay

[0083] To determine whether virus detected by LNAIA was attached between the wall and bottom of the nanopillar labyrinth, the substrate was freeze dried post-assay, followed by platinum deposition and imaging by SEM (JSM-7800F Prime, JEOL Ltd, Akishima, Japan).

Example 3. Confirmation of Formation of Supported Lipid Bilayer (SLB) on Surface of Nanopillar Array

[0084] The substrate generated in Example 1.4 by applying the small unilamellar vesicles (SUVs) (Example 1.1) onto the nanopillar-patterned substrate (Example 1.3) was observed via transmission electron microscopy (TEM) (120 kV, Talos L120C, FEI, Hillsboro, United States) and fluorescent microscopy (TE-2000, Nikon, Tokyo, Japan), and the obtained results are shown in FIG. 1B (h-n). As a result, the formation of SLB on the nanopillar array was observed.

[0085] SLB was designed to fully cover the side wall and top part of the nanopillars and the bottom of the substrate for efficient virus entrapment between the pillar wall and the bottom surface.

[0086] For characterization of the SLB coverage on a nanopillar substrate, the phosphate head groups of SLB were dyed by uranyl acetate, embedded in Spurr's resin, microsectioned via ultramicrotome, and observed via TEM (FIG. 1B (h) and (i), FIGS. 2 and 9). The dark line in FIG. 2 (i) reveals that the dyed supported lipid bilayer closely follows the nanopillar substrate surface.

[0087] Due to the high surface area of the nanopatterned array as compared to a flat substrate, the fluorescence intensity of the nanopatterned substrate was much stronger than is seen on a flat surface. The theoretical surface area ratio between the pillar-patterned and flat areas of the LNAIA kit prepared in Example 1.4 was calculated using following Formula 1, for comparison with the fluorescence intensity ratio. The obtained results are shown in FIG. 1B (k-m).

[00001]$\begin{array}{cc}\frac{A_{\mathrm{cell}}}{A}=1+\frac{2\mathrm{hp}}{R}=2.05& \left(\mathrm{Formula}1\right)\end{array}$

[0088] A.sub.cell is the total surface area of a unit cell containing a single cylindrical pillar, A is the surface area of a unit cell without a pillar, h is the height of a pillar (200 nm), p is the surface coverage of the pillars, and R is the radius of a pillar (100 nm).

[0089] This calculation yielded a ratio of 2.05, which closely matched the measured fluorescence ratio of 1.93, further indicating SLB formation along the nanopillar pattern (FIG. 1b (k-m)).

Example 4. Virus Detection by LNAIA

[0090] A fluorescence recovery (https://doi.org/10.1007/978-1-61779-207-6_26) was conducted after photobleaching (FRAP) experiments to investigate the fluidity of the SLB on a nanopillar substrate, and the obtained results are shown in FIG. 1n (n). A flat SLB kit prepared referring to Example 1.4 using a 4-inch glass wafer substrate without nanopillar array was used for comparison.

[0091] The degree of fluorescence recovery after bleaching indicates that SLB covers the nanopillar substrate without losing fluidity. In previous studies combining nanopatterned substrates and SLB, SLB either did not fully cover the nanopattern or formed a suspended structure due to either hydrophobic surface or unsuitable liposome size. In LNAIA provided in the present description, the nanopillar substrate was made of silica to achieve high hydrophilicity and proper dimensions, enabling homogeneous formation of a lipid bilayer along the pattern on the substrate (FIGS. 2 (a) and (b)).

[0092] Then, localization events of fluidic, fluorescent antibodies around the target virus were monitored via conventional epifluorescence microscopy. During the LNAIA assay described in Example 2, fluorescent images before and after a 20-minute sample loading were captured and shown in FIGS. 2 (c) and (d). As an H1N1 virion approaches the antibody-modified LNA, multiple polyclonal antibodies bind to the virus, localizing the fluorescent antibodies to an area roughly the size of a single virus. The concentrated antibodies generate a readily-detectable fluorescent spot corresponding to a single virus with a high signal to background noise ratio.

[0093] For quantitative virus detection, the increased number of fluorescence spots (ΔN) was automatically counted using a particle-counting software (MosaicSuite particle tracker plugin of image J). It was found that the nanopillar array structure plays an important role in increasing assay sensitivity and target capturing efficiency. The fast, strong colocalization of Cy3-antibody and immobilization of target virus resulted from the three-dimensional antigen-antibody interactions generate a high fluorescence signal to background signal ratio that enables efficient target detection by conventional fluorescence microscopy (FIGS. 2 (a) and (c)). On the other hand, the inefficient colocalization of Cy3-antibodies and high lateral mobility of viruses on flat SLB hamper visualization of single virus over a background, and there was nearly no difference in fluorescence signal before and after sample loading (FIGS. 2 (b) and (d)). Since the fluorescence-antibody on the substrate on which flat SLB is formed did not aggregate, the fluorescence spot cannot be observed with a conventional fluorescence microscope, and a high-end total internal fluorescence microscope (TIRFm, TE-2000, Nikon, Tokyo, Japan) is required for detecting single-molecule fluorescence-antibody signals.

[0094] In addition, after performing the LNAIA assay of Example 2, the substrate was freeze-dried, and the freeze-dried substrate was observed with SEM (Reference Example 3). The obtained image is shown in FIG. 2 (e). as shown in FIG. 2 (e), the position of virus on LNA is typically between the sidewall of a pillar and the bottom surface, further showing the importance of protruding nanopillar structures with a hexagonal array for efficient capture of viruses.

[0095] The LNAIA was performed using SLB with antibody (Ab-O) and SLB without antibody (Ab-X), and ΔN value obtained thereby was shown in FIG. 2 (f). As shown in FIG. 2 (0, in case of LNA (lipid nanopillar array), the ΔN value obtained in the test group with antibody was significantly larger than that of a control group without antibody (p-value of 0.0286), while in case of flat SLB, there is no significant difference between ΔN values of the test group with antibody and the control group without antibody (p-value of 0.028).

[0096] Trajectories of fluorescence spots on the nanopillar SLB observed via epifluorescence microscope were shown in FIG. 2 (g), and trajectories of fluorescence spots on flat SLB observed via TIRF microscope (TE-2000, Nikon, Tokyo, Japan) were shown in FIG. 2 (h). The trajectories of fluorescence spots shown in FIGS. 2 (g) and (h) indicate near-immobilization (LNA; FIG. 2 (g)) and free diffusion (flat SLB; FIG. 2 (h)) of virus particles for LNA and flat SLB surfaces, respectively. While the trajectories of localized fluorescence spots on LNA were observable via conventional epifluorescence microscopy, the trajectories of fluorescence spots on the flat SLB could not be detected via conventional epifluorescence microscopy and were only observable via TIRF microscopy (FIG. 2 (g-j)). The LNA boosts the binding kinetics of the virus to the surface antibodies due to the enlarged 3D surface area and multiple protrusions enhancing the collisions between surface and virus.

Example 4. Comparison of LNAIA and ELISA

[0097] The results obtained by performing LNAIA and ELISA referring to Example 2 and Reference Example 2, respectively, were shown in FIGS. 3b and 3c. In case of ELISA, nonspecific viruses, H3N2 and adenovirus (AdnV), were not differentiated from target virus (H1N1) at low concentration (at about 10.sup.5 particles/chamber or lower) (FIG. 3c). Although the same antibody was used for both LNAIA and ELISA, LNAIA showed significantly higher selectivity compared to ELISA (FIG. 3b).

[0098] Such high selectivity of LNAIA is due to the following hallmarks of LNAIA: Signal generation is accomplished by the cooperative interaction between the Cy3-antibody and the viral target. False positive signals (nonspecific bindings) are efficiently eliminated with LNAIA because true positive signals (specific bindings) are only produced when the concentration of Cy3-antibody exceeds the threshold of the fluorescence microscope, and each individual clustered spots can be reliably imaged and analyzed only when virus targets were multivalently captured by fluorescent antibodies (FIG. 3a). In addition, extracellular molecules in analytes cannot be bound by SLB, an artificial cell membrane without membrane proteins (indicating that SLB is an excellent surface with minimal nonspecific bindings). The above results can be further supported by lower background signals with the LNAIA negative control than with the ELISA-based negative control (FIG. 3c).

[0099] Importantly, the mean of dN (ΔN) values at 10.sup.5 section with LNAIA display a linear relationship. The detection limit of LNAIA is about 150 virus particles, which showed about 10,000-fold higher sensitivity than commercially available H1N1 ELISA. Moreover, LNAIA requires only 25 minutes while ELISA required 3 hours or more due to laborious antigen binding, labeling and washing procedures (FIG. 3c).

[0100] The examples of the present invention are described in detail as above. For those of ordinary skill in the art, it is clear that these detailed descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.