Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

Summary

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99^th percentile cutoff ∼10-400 pg/mL, depending on the assay).

Abstract

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.

Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm². The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

Introduction

Chemical sensors based on nanohole arrays have been a subject of numerous investigations since the first report on extraordinary optical transmission (EOT) was published by Ebbesen et al. in 1998¹. When light impinges on periodic arrays of nanohole structures of sub-wavelength dimensions, enhanced transmission occurs at specific wavelengths. This occurs when the incident light couples with Bloch-wave surface polariton (BW-SPP) and/or localized surface plasmons (LSP)².

The underlying physical principle exploited when biosensing with such periodic arrays is simple. Adsorption of molecules onto or near the interface of metal changes the dielectric constant of the medium in contact with the metal, in turn shifting the location of the transmission bands in the spectrum. The spectrum itself can be adjusted by nano-engineering the shape, size, and separation distance^3,4,5. By design, sensors based on EOT have characteristic bands in their spectra that facilitate specific assignments^6,7,8 during the investigation of molecular binding events. This is a crucial advantage over commercially available surface plasmon resonance (SPR) platforms.

Sensors using EOT typically involve a light source optically aligned such that a collimated beam is incident on the sensing surface. Techniques to generate large nanohole surfaces, such as co-polymer templates and interference and nanosphere lithography, have poor reproducibility⁹. Due to these limitations in accurately fabricating large surfaces that show the EOT phenomenon, an optical microscope was required to correctly position the light source and detector. To simplify the technique, high-quality nanoimprinting lithography (NIL)¹⁰ was employed. This enabled the production of large sensor surface areas¹¹ (mm-scale), removing the need for a microscope to look for the sensing surface on a chip. Instead, this sensor could be easily interfaced with a standard fiber optic cable.

Since the transmission peaks for this nanohole array are contained in the visible to near-infrared region (NIR), it is perfectly suited to sensing binding events for biomolecules in an aqueous environment. The expected optical behavior of the nanohole array was simulated. The result was then verified through studies with liquids of standard refractive indexes (RI). This array was then used to measure the concentration of cardiac troponin I (cTnI) in the complex background of human serum. cTnI is the clinical gold standard for the diagnosis of acute myocardial infarction.

Using this sensor, it is possible to detect and quantify cTnI in human serum at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. The detection is much quicker than the most commonly used technology in this domain, enzyme-linked immunosorbent assay (ELISA). Furthermore, the sensing surface can easily be regenerated and therefore reused. Hence, this work demonstrates the promise of nanohole arrays as a viable point-of-care (POC) technology for biosensing within complex biofluids.

Protocol

1. Fabrication of the Sensor and Acquisition of the Data

1. Preparation of the nickel mold
   1. Coat a 220 nm-thick layer of negative electron beam resist onto a 600 µm-thick 4-in silicon wafer. Write the designed nanohole array on this wafer using an electron beam lithography system.
      1. To accelerate the e-beam writing, write the patterns with a low dotmap (N) of 20k for each 300 µm field size (A) (i.e., there are 0.4 billion dots mapped on each 300 µm² area, and each dot will either be exposed by the e-beam or not, depending on the pattern design). Set the e-beam resist exposure dose for the resist to 110 µC cm⁻² and write at a current (I) of 800 pA.
         NOTE: In e-beam writing, the exposure dose (D) is controlled by the exposure time for each dot (T_dot), calculated by . For the exposure dose at 110 µC cm⁻², the e-beam dwelling time on each exposed dot is 0.5 µs¹². Since the array captures an area of 1.8 mm², there are a total of 36 patches of 300-µm² field areas stitched together to form one large, gold nanohole array.
   2. Develop the resist by immersing the 4 inch silicon wafer in the developer solution for 10 s and letting the wafer dry in air.
   3. Deposit a seed layer of a metal, such as nickel, copper, or aluminum, on the silicon wafer.
   4. Electroplate the wafer in a plating system in a nickel sulfamate bath. Carry out the electroplating in two steps. In the first step, lasting 95 min, use a current density of 0.7 A dm⁻²; this completely fills the nanopatterns with nickel. In the second step, lasting 125 min, use 12 A dm⁻² to reach 300 µm as the final nickel mold thickness (20 nm). Ensure that the pH value is at 3.5 - 3.8 and that the temperature is at 52 - 54 °C.
   5. Separate the nickel mold from the silicon substrate by applying gentle mechanical force. Soak the nickel mold in about 100 mL of positive photoresist removal reagent overnight to wash away the residue from the e-beam resist.
   6. Feed the nickel mold into an oven and dry it at 100 °C for 3 h. Clean it in a plasma etching system with O₂ gas at 10 sccm and 100 W for 3 min.
2. Fabrication of the gold nanostructure
   1. Coat 150 µL of heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane (FDTS) on the nickel mold in a self-assembly monolayer (SAM) coating machine at 80 °C.
      NOTE: This will form an anti-adhesive layer, which will enable the separation of the mold from the photoresist ("demolding") after the completion of the nanoimprinting step. The vaporizing time should be 180 s, and the reaction time should be 900 s.
   2. Imprint the nanopatterns on a 4-in glass wafer that has been coated with a 300 nm-thick layer of photo-curable NIL resist using a nano-imprinter at a pressure of 10 bar and a temperature of 40 °C for 10 min.
   3. Transfer the mold, the photoresist, and the glass wafer to a UV light curing system and photocure with 75 mW cm^-2 of UV exposure for 30 s.
      NOTE: If all steps have been followed correctly, the nickel mold should easily be demolded from the photoresist.
   4. In a reactive ion etching (RIE) system, perform a blank etch of the photoresist on the glass substrate, with an O₂ gas flow of 10 sccm, at 50 W for 2 s to expose the glass on the indented areas.
   5. Deposit a 5 nm-thick layer of chromium (Cr) for metal adhesion and a 100-nm layer of gold (Au) for the plasmonic sensor on the glass wafer in an electron-beam deposition machine. Use a deposition rate of 1 Å s⁻¹ for Cr and 2 Å s⁻¹ for Au.
   6. Perform lift-off of the photoresist by O₂ plasma etching for 3 min followed by a 15-s sonification step in acetone.
   7. Dice the sample into 5 mm × 5 mm chips. The nanohole array will occupy the central 2 mm × 2 mm of the chip.
3. Acquisition of the data
   1. Set up the apparatus to make the optical measurements such that a beam of white light exiting through the end of the transmitter optical fiber is collimated and is incident on the sensor surface (nanohole array) at 90°.
      NOTE: Light is transmitted through the whole nanohole array.
   2. Collect the transmitted signal with the receiver optical fiber and record it with a UV-visible spectrometer operating within the range of 300 to 1,000 nm.
   3. Set the acquisition time for each frame to 20 ms. Average 100 frames to obtain the final spectrum to lower the noise in the measurements.
   4. Use plotting software to analyze the data based on the previously identified transmission peaks (using a Lorentz-based method).

2. Sensor Bulk Sensitivity Test

1. Deposit the standard RI liquid into the liquid cell, with the RI varying from 1.31 to 1.39.
2. Immerse the sensor chip in the standard RI liquid and align it with the beam of white light. Obtain the transmission spectrum.
3. Clean the sensor chip after each measurement with a surface-active cleaning reagent and dry it with nitrogen gas.

3. Sensor Surface Modification

1. Prior to any chemical modifications, clean the sensor chips by sequential immersion in isopropanol, acetone, and deionized water. Dry at room temperature in a stream of dry nitrogen gas.
2. Incubate the sensor chips in an ethanolic solution of 0.4 mM 10-carboxy-1-decanethiol and 1.6 mM 1-octanethiol for 12 h at room temperature.
   NOTE: This will form an amine-reactive self-assembly monolayer (SAM).
3. Use ethanol to rinse thoroughly and dry at room temperature.
4. Make a mixture of 75 mM sulfo-N-hydroxysuccinimide (sulfo-NHS) and 15 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Immerse the chips in this mixture for 15 min.
   NOTE: This will activate the carboxylic group of the SAM.
5. Spot 50 µL of 200 µg/mL anti-troponin antibody solution made in a pH 4.5 acetate buffer on the sensor surface and incubate for 30 min.
6. Deactivate the unreacted esters by immersing the sensor chip in 1 M ethanolamine-HCl solution for 15 min.
7. Rinse the chip with deionized water and dry it in a stream of dry nitrogen gas at room temperature.

4. cTnI Assay

1. Block any non-specific binding by spotting 100 µL of 1% bovine serum albumin (BSA) solution onto the surface. Incubate for 15 min.
2. Rinse the sensor chips three times in phosphate-buffered saline (PBS) solution. Insert the chip into the measurement cell to record the transmission spectrum.
   NOTE: This is the reference spectrum.
3. Spot 50 µL of cTnI standard onto the chip surface and incubate in a moist environment for 30 min.
4. Rinse the sensor chips three times in PBS solution and insert it into the measurement cell to record the transmission spectrum.
   NOTE: This is the after-binding spectrum.
5. Submerge the chips in 50 mM glycine-HCl (pH 2) for 1 min and then rinse in PBS solution three times to regenerate the chip surface. Measure the transmission spectrum in PBS to verify the success of the regeneration step.

5. Surface Plasmon Resonance (SPR) Measurement

1. Run the multiplexed SPR sensor chip on the SPR system with PBS-T buffer.
   NOTE: The composition of PBS-T buffer is 20 mM Na-phosphate, 150 mM NaCl, and 0.05% Tween 20. The pH is 7.4.
2. Use cTnI standard and the antibody, as described in step 4.
3. Activate 3 out of the 6 available channels with a mixture of EDC (0.2 M) and sulfo-NHS (0.05 M) for 5 min. Perform a 5 min injection of 50 µg/mL antibody 560 and a 5-min injection of 1 M ethanolamine-HCl solution.
4. Rotate the sensor chip by 90° and inject the cTnI standards at different concentrations (75, 30, 7.5, and 2.5 ng/mL).
5. Observe the conjugation to the antibody at spots of interaction on the chip in real time through the SPR readout.
6. Regenerate the chip by injecting 50 mM glycine-HCl (pH 2) for 1 min.

Results

The optical setup for taking measurements is shown in Figure 1A. An image of the actual nanohole array is given in Figure 1B. To understand the physics driving the sensing process, the COMSOL simulation software was used to simulate the distribution of the plasmonic field in an aqueous environment. The results from the simulation were then related to the actual measurement. A previously published study contains details of the ass...

Discussion

Simulating the interaction between incident light and the nanostructures makes it possible to identify the appropriate peak (in the transmission spectrum), whose shift can be recorded as a function of the concentration of the analyte. It is important to note that the localization of the bands with respect to the structure of the sensor is crucial to the choice of the right band, whose shift can be tracked to sense the analyte. The visualization can be achieved through simulations. This is also critical to the design of a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Electron Beam Lithography setup	Elionix ELS 700
o-Xylene	Sigma Aldrich	95662
EB resist	Sumitomo	NEB-22A2
Developer reagent	Shipley Company	Microposit MF 321
Electroplating machine	Technotrans AG RD 50
Photoresist stripper	Rohm and Haas Electronic Materials LLC	Microposit Remover 1165
Etching System	Trion Phantom
Heptadecafluoro-1,1,2,2-tetrahydrodecyl)trichlorosilane	Gelest (PA, USA)	78560-44-8
SAM coater	Sorona Inc.	AVC 150M
Photo-curable NIL resist	micro resist technology GmbH	mr-UVCur21-300nm
Light Curing System	Dymax	Model 2000 Flood
E-beam deposition machine	Denton Explorer
UV-visible spectrometer	Ocean optic HR2000+ (Dunedin, FL, USA)
Standard refractive index liquids	Cargill Inc (Cedar Grove, USA)	18032
Plotting software	Origin	Origin Pro 9
10-carboxy-1-decanethiol	Dojindo Laboratories (Japan)	C385-10
1-octanethiol	Sigma-Aldrich, MO, USA	471386
Sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide	BioRad (CA, USA)	1762410
Anti-troponin antibody 560	Hytest (Finland)	4T21
Ethanolamine-HCl solution	BioRad (CA, USA)	1762450
Surface Plasmon Resonance setup	BioRad XPR36 (Haifa, Israel)
Multiplexed SPR chip	BioRad	GLC
Human cTnI standard	Phoenix Pharmaceuticals	EK -311-05
Glycine-HCl	BioRad (CA, USA)	1762221