Ultrasensitive Detection of Bacterial Protein Toxins on Patterned

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Ultrasensitive detection of bacterial protein toxins on patterned microarray via SPR imaging with signal amplification by conjugate nanoparticle clusters Alexander Lambert, Zhanjun Yang, Wei Cheng, Zhenda Lu, Ying Liu, and Quan Cheng ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00260 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Ultrasensitive detection of bacterial protein toxins on patterned microarray via SPR imaging with signal amplification by conjugate nanoparticle clusters

Alexander Lambert1, Zhanjun Yang1, Wei Cheng1, Zhenda Lu3, Ying Liu2,*, Quan Cheng1,*

1

Department of Chemistry, University of California, Riverside, California, 92521, USA 2 3

Department of Chemistry, Nanjing University, Nanjing 210023, China

College of Engineering and Applied Science, Nanjing University, Nanjing 210023, China

*

Corresponding authors: Quan Cheng

Tel: (951) 827-2702; Fax: (951) 827-4713; Email: [email protected] and Ying Liu Tel: +86-25-89681918; Email: [email protected]

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ABSTRACT

Sensitive detection and monitoring of biological interactions in a high throughput, multiplexed array format has numerous advantages. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy and SPR imaging via the effect of accumulation of conjugated nanoparticles of varying sizes. Bacterial cholera toxin (CT) was chosen for the demonstration of enhanced immunoassay by SPR. After immobilization of CT on a gold surface, specific recognition is achieved by biotinylated anti-CT. The signal is amplified by the attachment of biotinylated 20nm AuNP via streptavidin bridge, followed by attachment of 5nm streptavidin-functionalized Fe3O4NP to the AuNP-biotin surface. The continuous surface binding of two differently-sized conjugated nanoparticles effectively increase their packing density on surface; significantly improve SPR detection sensitivity, allowing quantitative measurement of CT at very low concentration. The dense packing of conjugated nanoparticles on the surface was confirmed by atomic force microscopy characterization. SPR imaging of the immunoassay for high-throughput analysis utilized an Au-well microarray that attenuated the background resonance interference on the resulting images. A calibration curve of conjugated nanoparticle binding signal amplification for CT detection based on surface coverage has been obtained that shows a correlation in a range from 6.31 x 10-16 to 2.51 x 10-13 mol/cm2 with the limit of detection of 5.01 x 10-16 mol/cm2. The absolute quantity of detection limit using SPR imaging was 0.25 fmol. The versatile nanoparticles and biotin-streptavidin interaction used here should allow adaptation of this enhancement method to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules.

Keywords: Surface plasmon resonance, SPR imaging, nanoparticle amplification, microarray, plasmonic microchip


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Surface plasmon resonance (SPR) biosensors have become an indispensable tool for studying biomolecular interactions in pharmaceutical and biomedical research. Capable of real-time and end-point detection that can be label-free and highly sensitive, SPR allows for measurement of analyte concentration and binding kinetics in a fast, convenient, and nondestructive fashion. The Kretschmann configuration is the most widely employed physical setup for SPR, which utilizes a thin (approximately 50 nm) layer of a noble metal (usually gold) deposited on a glass substrate that is attached to a prism1. Measuring the optical characteristics of light reflected from the prism, including resonance angle, intensity, phase, and polarization, provides information about the biomolecular interactions taking place on the surface2-4. Examples of use include protein conformation studies5, biomarker profiling6, aptamer selections7, and antibody selections8, which have produced high-affinity ligands that recognize specific protein targets. While highly useful for determining a variety of important kinetic and affinity parameters of biological interactions, one major drawback of SPR is its low throughput9. This problem has been largely circumvented with the advent of SPR imaging. Rather than the scanning-angle or scanning-wavelength measurements commonly employed in SPR spectroscopy, SPR imaging generally measures at a fixed angle. Differences in reflectivity are monitored over time, which provides spatial capabilities of imaging and allows for multiplexed detection and high-throughput bioanalysis10. By using a CCD camera for signal detection, the images of the chip can be recorded allowing simultaneous analysis of many interactions. SPR imaging has been reported for the bioaffinity detection of a wide range of analytes, including proteins11,12, nucleic acids13-15, carbohydrates16, Escherichia coli bacteria17, and receptor-guest interactions18. There are multiple directions of development in the fabrication of the arrays for SPR imaging research. Microfluidics (Glass/PDMA biomolecular screening chip) offer the distinct advantage of possible individual addressability, which enables multiple chemical environments to be explored in a multiplexed fashion19,20. However, microfluidics fabrication requires complicated and precise design of flowcells. Contact printing has also been widely used with solid pins, which enables easy deposition of viscous solutions, reproducible and efficient printing, and uses a simple cleaning procedure21-23. Effective passivating reagent is necessary after microarray printing to avoid nonspecific adsorption for unarrayed areas24. ACS Paragon Plus Environment

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SPR imaging analysis is generally displayed through differential sensorgrams25 or rendered false color images with background subtraction/correction26-28. During measurement, surface plasmon resonance occurs across the entire surface, giving rise to significant background signals. As the background signal varies in response to surrounding light excitation and solution conditions, the sensitivity and the accuracy of the measurements in the targeted area can be severely compromised with the fixed background analysis. Some efforts have been made to reduce the background resonance by nonchemical approaches such as the use of a patterned SPR-carrying layer to obtain metal spots or islands separated by uncoated glass19,29,30. Here we used a novel SPR imaging microarray fabricated earlier in our group31, wherein a second layer of 100 nm Ti was deposited on gold sublayer via photolithography to generate Au-well microarray. The spatial variation of the metal thickness restricts the excitation of surface plasmons in the desired patterns and attenuates the evanescent field in the background area, mitigating the possible background interference. Recent developments of nanomaterials have significantly enhanced the detection sensitivity of biosensors. AuNP has been reported to improve the sensitivity of end-point type of SPR detection for biomolecules such as progesterone32, DNA, and protein33, but the enhancement is limited. Packing density of nanoparticles is important to achieve satisfactory SPR signal amplification. However, the rigid structure of large nanoparticles, such as Au, resists close packing and leave vacant areas that do not receive the enhancement effect, impairing amplification. To increase the packing density of AuNPs on a surface, Corn et al. has reported the use of a poly(A) polymerase extension reaction coupled with poly(T)-coated AuNP to amplify signals from surface-bound miRNA34. To sufficiently occupy the space among bound AuNPs, polymer was grown on the AuNP surface to further amplify SPR signal for protein detection35. Using smaller-sized nanoparticles for signal enhancement in bioassays is less common, because it is difficult to achieve enough packing density for the small particles to be effectively sensed by a general technique36. Here we combine the approaches, and connect two differently-sized nanoparticles together to enhance packing density on surface for SPR signal amplification of cholera toxin (CT) protein detection. For experimental convenience, protein CT was covalently immobilized on plain gold substrate and Au-well microarray to demonstrate the principle. After surface blocking with BSA and ACS Paragon Plus Environment

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immuno-attachment of biotin-antiCT and streptavidin, 20 nm biotin-functionalized AuNPs were applied. The role of AuNP-biotin was twofold: to increase SPR detection signal and to act as building blocks to generate space for subsequent Fe3O4NP filling. 5nm streptavidin-functionalized Fe3O4NP was incubated on the surface and attached to biotin functionalized AuNP via biotin-streptavidin interaction; the FeNPs occupied the gap area between AuNPs and further enhanced the SPR signal. The amplification method relied on the accumulation effect of differently-sized conjugated nanoparticles, which efficiently utilize the space and significantly enhance packing density. Given the many and varied types of nanoparticles and wide availability of biotinylated antibodies for specific biomolecules, this method should be broadly applicable to almost any protein of interest.

EXPERIMENTAL

Materials and Reagents. Streptavidin and (+)-Biotinyl-3,6,9,-trioxaundecanediamine (BA) were obtained from Thermo Scientific (Rockford, IL). N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 2-(2’-aminoethoxy) ethanol

(AEE),

polyoxyethylene

(20)

sorbitan

monolaurate

(Tween

20),

16-mercaptohexadecanoic acid (16-MHDA), cholera toxin (CT), 11-mercaptoundecanoic acid (MUA), Bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO). Biotinylated rabbit anti-cholera (biotinylated antiCT) serum was from ViroStat (Portland, Maine). All proteins solutions were prepared in 20 mM phosphate buffered saline (PBS) (containing 150 mM NaCl, pH 7.4) unless indicated otherwise. Citrate-stabilized AuNP solution with particle size of about 20 nm was prepared according to the literature37 and stored in brown glass bottles at 4 oC, which is stable for at least 1 month. Poly(acrylic acid) (PAA) wrapped Fe3O4NP solution with particle size of about 5 nm was prepared according to the literature38, which is also stable for at least 1 month.

Instrumentation. A dual channel SPR spectrometer NanoSPR-321 (NanoSPR, Addison, IL) with a GaAs semiconductor laser light source (λ=670 nm) was used for all SPR spectroscopy measurements. The device comes with a high-refractive index prism (n=1.61) and 30 µL flow ACS Paragon Plus Environment

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cell. Surface interaction and modification were monitored using the angular scanning mode around the minimum angle. SPR imaging was conducted using a home-built, previously reported instrument setup.39 Au-well microarray chips were mounted on a home-built optical stage housing a 300 µL flow cell. An equilateral SF2 triangular prism (n = 1.61) was then put in contact with the glass substrate with a matching liquid (n = 1.61). The optical stage was fixed to a rotating goniometer that allows the tuning of the incident angle of a red light (648 nm) emitting diode (LED) that excited the surface plasmons on the metal surface. The reflected images of the microarray were captured by a cooled 12-bit CCD camera (QImaging Retiga 1300) with a resolution of 1.3 MP (1280×1024 pixels) and 6.7 µm×6.7 µm pixel size using p-polarized light. Difference images were obtained by digitally subtracting one image from another. All the experiments were carried out at room temperature (23 oC). Atomic force microscopy images were obtained using a Veeco Dimension 5000 atomic force microscope (Santa Barbara, CA) with manufacturer-provided software. All images were obtained in tapping mode.

Preparation of functionalized nanoparticles. AuNP was modified with hydroxyl and biotin groups according to a literature protocol40 with some modification. In brief, a 0.80 nm AuNP dispersion (in citrate) was gently mixed in equal volume with 1.82 mg/mL Tween 20 (in 10mM PBS), which then stood for 30 min to allow physisorption to occur. 0.5mM 16-MHDA solution was added and stood for 3 hr to allow chemisorption, which was followed by three sets of centrifuging (30 min at 15700 g) and resuspension (in 10 mM PBS with 1.82 mg/mL Tween 20). The resulting 16-MHDA-modified AuNPs were then reacted with 50mM NHS/200mM EDC (in 10mM PBS) for 5 min. After washing with 10mM PBS (with 1.82 mg/mL Tween 20, 5 min centrifuge), the remaining NHS ester-alkanethiol modified AuNP were reacted with an aqueous solution of 22 mM AEE and 2.4 mM BA for 20 min. After again washing with PBS, the resulting AuNP-hydroxyl-biotin (hereafter referred to as AuNP-biotin) was stored at 4 oC. Fe3O4NP-streptavidin was prepared first by activating the carboxylate groups of the Fe3O4 nanoparticle surface by reacting with a mixture of freshly prepared 100 mM NHS and ACS Paragon Plus Environment

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400 mM EDC solution (10 mM PBS, pH 7.0) for 5 min. Then, after washing with 10mM PBS, the Fe3O4NP were incubated with 1.6 mg/mL streptavidin solution overnight. After washing with 10 mM PBS, the resulted Fe3O4NP-streptavidin was stored at 4 oC.

Au surface preparation for immunoassays. SPR spectroscopy gold sensor chips were fabricated in a cleanroom via E-beam evaporation, onto cleaned BK-7 glass slides, of a 2 nm thick chromium adhesion layer, followed by deposition of a 46 nm thick gold layer. Au-well microarray was fabricated according to our reported protocol31 with modification. As shown in Figure 1, a BK7 glass substrate was used for the fabrication with E-beam evaporation of 2 nm titanium as the adhesion layer and of 48 nm gold as the SPR active layer. A photoresist AZ5214E was then spin-coated and patterned by photolithography. A second E-beam evaporation was performed to deposit 100nm titanium on the patterned substrate. The photoresist was then removed with acetone. After rinsing with ethanol and water, the obtained Au-well microarray chips were stored under vacuum before use.

SPR signal amplification with AuNP-biotin and Fe3O4NP-streptavidin for CT detection. The gold chip for SPR spectroscopy and Au-well microarray chip for SPR imaging were incubated in 1mM MUA ethanol solution for 18 h to form a self-assembled monolayer with carboxyl functional group on the surface. After extensive rinsing with copious ethanol and DI water, the chip was dried under a N2 stream. To activate the carboxyl acid group, EDC (400 mM)/NHS (100 mM) solution was injected into the flow cell and incubated for 30 min followed by 10 min surface rinsing. Each subsequent step in the immunoassay was also followed by a 10 min rinse. Varied concentrations of CT were injected subsequently and incubated for 30 min to allow formation of covalent amide linkages, which was followed by a 10 min rinsing to eliminate any residual CT in solution. Passivation of the unused activated carboxyl groups was preformed by incubation with 1mg/mL BSA solution for 30 min. The immunoassay binding was carried out with injection of 0.25 mg/mL biotinylated anti-CT and 30 min incubation. Following this, 0.5 mg/mL streptavidin was injected and incubated for 30 min to prepare the surface for conjugated nanoparticle binding. The amplification with conjugated nanoparticles attachment was conducted by injection of ACS Paragon Plus Environment

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AuNP-biotin solution and 10 min incubation. After 10 min surface rinsing to remove nonspecific and unstable surface bound AuNP-biotin, Fe3O4NP-streptavidin was injected subsequently and incubated for 10 min to further enhance SPR signal. The surface was rinsed with water for 10 min to remove unstable surface bound Fe3O4NP-streptavidin.

RESULTS AND DISCUSSION

SPR signal amplification with AuNP-biotin and Fe3O4NP-streptavidin binding. AuNP has been widely used to enhance detection signal for SPR biosensors41,42. However, the AuNP have rigid structure that limits its packing density on the surface, so the full extent of amplification is not realized. To overcome this problem, we looked to fill in the space with another type of nanoparticle. Fe3O4 has been used previously in a signal enhancing capacity, but not extensively, since it is too small on its own to effectively enhance the signal36,43. As a result, surface enzyme polyadenylation reaction was applied to generate poly(A) tail to increase the binding positions of T30 coated AuNP34, and atom transfer radical polymerization (ATRP) reaction was initiated from AuNP surface to fill the packing vacancy35. Magnetic nanoparticles have been frequently used in bioseparation and for analyte purification and enrichment44, but there are few reports about their applicability in bioassays due to their extremely small size so that sensitive detection is difficult to achieve using a general technique36,43. Here, 20 nm AuNP-biotin was initially applied on the surface as a building block to create large spatial positions and surface area for binding. 5 nm Fe3O4NP-streptavidin were then accumulated around AuNP via the strong biotin-streptavidin interaction, causing the Fe3O4NPs to occupy the gap areas between the AuNP. This increased the packing density on the surface for the purpose of amplifying the signal change in SPR detection. The high specificity of the biotin-streptavidin interaction brings Fe3O4NPs selectively to the surface-bound AuNP, minimizing effects on the binding signal due to nonspecific adsorption. The SPR signal enhancement was demonstrated with bacterial cholera toxin. As displayed in Figure 2, for simplicity of the experiment, CT is covalently linked to surface via amide bond to demonstrate the feasibility of the overall amplification process. To eliminate nonspecific adsorption, the free activated carboxyl acid groups of MUA ACS Paragon Plus Environment

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on the surface that were not attached to CT were blocked by incubation of 1mg/mL BSA solution for 30 min. The presence of attached CT on the surface was initially recognized by injection and bonding of 0.25 mg/mL biotinylated anti-CT for 30 min. This was followed by injection of 0.5 mg/mL streptavidin to specifically bind to the exposed biotin tag. The captured streptavidins left additional binding sites available for further binding via incubation of AuNP-biotin. SPR resonance angle shifted 0.59° upon AuNP-biotin binding, and the signal was further increased 1.48° for Fe3O4NP-streptavidin incubation (Figure 3a). The combination of AuNP-biotin and Fe3O4NP-streptavidin attachment (2.07°) amplified the SPR degree shift to about 40 times more than the degree shift from the initial injection of 0.1 µM CT (0.05°). In the control channel, PBS buffer was injected instead of CT under the same conditions, followed by surface blocking with BSA and surface incubation with biotinylated antiCT, streptavidin, AuNP-biotin and Fe3O4NP-streptavidin sequentially. There was no measurable SPR signal change for biotin-antiCT, streptavidin incubation and little SPR signal increase for AuNP-biotin incubation, demonstrating good specificity of signal enhancement. Unbound Fe3O4NP-streptavidin was not completely removed by surface rinsing due to its small size, which resulted 0.10° from nonspecific adsorption (Figure 3b), but this signal increase is negligible compared with substantial specific binding signal (1.48°, Figure 3a). To demonstrate the contribution of AuNP-biotin to Fe3O4NP-streptavidin accumulation, Biotin-BSA was immobilized on surface and the signal from direct binding of Fe3O4NP-streptavidin

to

Biotin-BSA

was

compared

to

the

binding

signal

of

Fe3O4NP-streptavidin in the presence of AuNP-biotin (Figure 3c). In the absence of AuNP, biotin-BSA only provided limited surface area for Fe3O4NP-streptavidin attachment, resulting 0.74° of SPR signal increase. With streptavidin as a connection bridge and AuNP-biotin as building blocks, Fe3O4NP-streptavidin had a larger effective surface area to bind and generated 1.39° of SPR signal increase. This result confirms the initial hypothesis that the AuNP-biotin attached on the surface increases spatial positions for Fe3O4NP-streptavidin accumulation, which further amplifies binding signal of Fe3O4NP-streptavidin by nearly a factor of two.

Morphological characterization of Fe3O4NP-streptavidin and AuNP-biotin nanoparticle ACS Paragon Plus Environment

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conjugate. The physical accumulation of Fe3O4NP-streptavidin around AuNP-biotin was characterized by AFM, which can provide useful topographic information for surface immobilized nanoparticles. Figure 2e shows the AFM image of the surface features of the adsorbed Fe3O4NP-streptavidin and AuNP-biotin with 0.1 µM CT immobilization. Unlike individual dots isolated on surface for sole AuNP binding45, here densely-packed block conjugate was observed on the surface in networks of small particles, resulting in larger islands with thickness above 30 nm and average feature size over 100 nm. The average size of individual small particle is about 10 nm, which approximates the sum of Fe3O4NP (5 nm)30 and streptavidin (3-5 nm)46. This confirms that Fe3O4NP-streptavidin accumulated around AuNP-biotin, fully occupying the exposed AuNP surface area and significantly enhancing nanoparticle packing density on surface.

Highly sensitive detection of protein toxin. CT is responsible for the deleterious effects of cholera infection, one of the most severe illnesses in developing countries. The lethal dose for cholera toxin in human is relatively low (LD50 = 250 µg kg-1), so there is a clinical need for accurate detection of CT with high sensitivity47, and ultrasensitive detection of CT has drawn considerable interest in recent years48. To demonstrate the overall sensitivity of the Fe3O4NP -streptavidin and AuNP-biotin SPR signal enhancement, CT was covalently immobilized on surface in varying amounts. Figure 4 shows a plot of the SPR signal increase after AuNP/Fe3O4NP enhancement as a function of CT surface coverage, where the surface coverage of CT was calculated based on our formerly reported method49. The conjugated nanoparticle amplified signal shows two segments of linear correlation to CT concentration. When surface immobilized CT amount is between 2.51x10-13 and 7.94x10-13 mol/cm2, the large protein amount generated substantial signal increase based on conjugated nanoparticle binding, resulting in a much steeper calibration curve than at lower concentrations. This type of segmental calibration had been observed by our previously reported method for CT detection49 and by others for IgG fluorescence detection50. The other linear range is from 6.31x10-16 mol/cm2 to 2.51x10-13 mol/cm2 (R2=0.932), which is more relevant for the desired range of ultrasensitive detection. Even at 6.31x10-16 mol/cm2 (corresponding to CT concentration of 4.52 pM), the immobilized CT still generated discernible SPR signal change ACS Paragon Plus Environment

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after conjugated nanoparticle binding. This concentration is much lower than most previously reported CT detection methods, including SPR signal amplification with ATRP polymer growth49, colorimetric method51, fluorescently labeled microarray52, and electrochemical biosensors that rely on lipid bilayer membranes supported on glass fiber53 or with a microfluidic technique54. The detection limit at 3σ was 5.01x10-16 mol/cm2, giving an absolute quantity detection limit of 0.25 fmol, which is comparable to a highly sensitive enzyme-catalyzed enhanced chemiluminescence reaction55.

SPR Imaging characterization of Au-well array and signal amplification measurement. The SPR imaging experiments were carried out using a home-built instrument arranged in the Kretschmann configuration40. The reflectance from the array was imaged with a CCD camera and the change in reflectance was recorded in real-time. Contact printing48,56, microfluidic cross-patterning with two PDMS chips57 and automatic arrayers58 have been used to generate protein arrays on flat gold substrates for SPR imaging. However, an effective blocking method is required to deactivate the unbound area to SPR and prevent background resonance and nonspecific adsorption from interfering with the images. Recently, we developed a microarray of 800 µm diameter gold wells surrounded by raised titanium platforms that attenuate the background evanescent field in the areas in between the wells31. As demonstrated in Figure 5a, surface plasmons only resonated and absorbed light in the circular Au well areas, darkening those areas, whereas the boundary titanium areas do not show any effect from the s-polarized light. With the immobilization of 1.29x10-12 mol/cm2 CT, the discussed immunoassay binding of biotin-antiCT, streptavidin and conjugated nanoparticles significantly increased surface refractive index in the Au wells (Figure 5b). For clearer results and easier analysis, a difference image is taken between pre- and post assay (Figures 5a and 5b, respectively), and the high reflection intensity in Figure 5d from conjugate nanoparticle amplification clearly demonstrates CT immobilization. By comparison, a difference SPR image was captured for 1.29x10-12 mol/cm2 CT without antibody binding and conjugate nanoparticle signal enhancement, and the CT immobilization signal was barely observed (Figure 5c). The corresponding 3D (Figure 5e) and 2D (Figure 5f) profiles were generated for the array elements from Figure 5d, demonstrating the high reproducibility across the array ACS Paragon Plus Environment

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and clean background. SPR imaging quantitative analysis of different concentrations of CT is shown in Figure 6. The displayed images are the difference images, and each image was a 2 x 2 array for CT concentration ranged from 3.16 x 10-15 to 1.29 x 10-12 mol/cm2. Reflection intensities after conjugated nanoparticle signal enhancement are listed. Figure 6a is a control array with no CT where the surface was blocked by BSA and subjected to the same experimental conditions for immunoassay binding and conjugate nanoparticle signal amplification. The control only showed a small signal (RI = 803±52.3 au), suggesting the conjugated nanoparticle binding signal enhancement has high specificity to CT. The reflection intensity difference also exhibited pronounced response to increasing CT concentration. The highest signal amplification is obtained with 1.29x10-12 mol/cm2 CT immobilization (RI = 16540±216.6), while 3.16x10-15 mol/cm2 CT immobilization generated signal increase of 1667±107.2. Reflection intensity plotted against CT surface coverage demonstrates a linear relationship from 3.16x10-15 to 1.29x10-12 mol/cm2, and using 3 S/N cutoff, the detection limit was determined to be 2.0x10-15 mol/cm2. Taking into account the individual Au-well surface area, the absolute quantity of detection limit is 3.2 amol, which is much lower than ultrasensitive electrochemical detection58 and fluorescence detection with signal amplification by fluorescent nanoparticles59, and 105 times more sensitive than direct CT detection on Au-well microarray via SPR Imaging.60 With the choice of appropriate biotinylated antibodies, this method can be applied to high-throughput detection of various proteins.

CONCLUSION We report here a novel SPR signal amplification strategy for highly sensitive detection of proteins by stacking of differently-sized nanoparticles. Immunoassay binding of biotin-antibody to the target protein and subsequent attachment of streptavidin is initially accomplished on gold substrate. This is followed by SPR signal amplification by two consecutive nanoparticle bindings of 20 nm AuNP-biotin and 5 nm Fe3O4NP-streptavidin, which greatly increases packing density on the surface and thus enhances SPR signal substantially. The immobilized CT has been detected in the range of 6.31x10-16 to 2.51x10-13 mol/cm2 and has a detection limit as low as 6.31x10-16 mol/cm2 (0.25 fmol). Additionally, ACS Paragon Plus Environment

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SPR Imaging detection of the amplified immunoassay was carried out using a novel Au-well microarray of 800 µm diameter gold wells surrounded by raised titanium platforms for attenuation of background evanescent field interference. The evanescent field is attenuated in the background area and SPR resonance is restricted in gold wells, therefore avoid background resonance interference and nonspecific adsorption. CT detection from the Au-well microarray demonstrated a linear relationship from 3.16x10-15 to 1.29x10-12 mol/cm2 with a detection limit of 2.0x10-15 mol/cm2 and an absolute quantity of detection limit of 3.2 amol. With various nanomaterials and biotin antibodies, this method can be well applied to high throughput detection of a wide range of proteins.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], Phone/Fax: 1-951-827-2702 (Q.C.). *E-mail: [email protected], Phone/Fax: +86-25-89681918 (Y.L.). ORCID Quan Cheng: 0000-0003-0934-358X Ying Liu: 0000-0001-5718-7804 NOTE The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Science Foundation (CHE-1413449). YL gratefully acknowledges the National Natural Science Foundation of China

(21605083,

21635005),

Natural

Science

Foundation

of Jiangsu

Province

(BK20160644), and the National Research Foundation for Thousand Youth Talents Plan of China.


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FIGURE CAPTION

Fig. 1. Schematic of the fabrication of Au-well arrays for surface plasmon resonance imaging.

Fig. 2. Sequence of surface modifications for SPR signal amplification: (a) immobilization of cholera toxin (b) addition of biotin-antiCT and streptavidin, (c) attachment of AuNP-biotin to streptavidin, and (d) binding of Fe3O4NP-streptavidin to AuNP-biotin. (e) AFM image of Fe3O4NP-streptavidin and AuNP-biotin conjugate nanoparticles binding on sensorchip in the presence of 2.51x10-13 mol/cm2 CT.

Fig. 3. SPR sensorgrams for (a) immunoassay of 2.51x10-13 mol/cm2 CT with AuNP-biotin and Fe3O4NP-streptavidin nanoparticle for signal amplification, (b) the control channel in the absence of CT, and (c) Fe3O4NP-streptavidin nanoparticle binding on covalently immobilized Biotin-BSA in the presence and absence of AuNP-biotin nanoparticles and streptavidin bridge.

Fig. 4. SPR response as a function of CT surface coverage after AuNP-biotin and Fe3O4NP-streptavidin conjugate nanoparticle amplification with streptavidin bridge.

Fig. 5. Raw SPR images of Au-well microarray (a) before CT immobilization and (b) after nanoparticles amplification of 1.29x10-12 mol/cm2 CT; difference SPR images of 1.29x10-12 mol/cm2 CT (c) before and (d) after nanoparticle amplification; (e) 3D and (f) 2D intensity profiles for the array elements of (c).

Fig. 6. Reflectivity as a function of CT surface coverage on microarray after Fe3O4NP-Streptavidin and AuNP-Biotin conjugate nanoparticle amplification.; Inset: SPR images of corresponding coverages.


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Figure 1


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Figure 2


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Figure 4


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Figure 6


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Ultrasensitive Detection of Bacterial Protein Toxins on Patterned

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