Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis
March 7th, 2018
In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.
The overall goal of this procedure is to amplify the signals of nanoparticle-based probes in assays carried out on either paper or glass supports. Using this method, you can acquire a colorimetric signal for spots that contain as few as 10 nanoparticles. The main advantage of this technique is its versatility.
It can be applied without major modification to different nanoparticles on paper, glass, and potentially other supports. To begin the procedure, prepare at least 10 milliliters of a 10 millimolar MES solution in deionized water. Adjust the pH of the solution to six with a four-molar sodium hydroxide solution.
Next, combine one milliliter of a 1.2 times 10 to the minus ninth molar suspension of 40 nanometer gold nanoparticles with one milligram of alpha mercapto omega carboxy PEG and vortex the mixture. Adjust the pH of the mixture to 12 with a four molar sodium hydroxide solution. Gently stir the mixture at room temperature overnight.
Then, centrifuge the mixture at 3, 800 times G for 15 minutes and discard the supernatant. Resuspend the pellet of PEG-modified gold nanoparticles in 500 microliters of deionized water by gentle pipetting. Next, dissolve five micromoles of EDC in 250 microliters of the 10-millimolar pH six MES buffer.
Dissolve 7.5 micromoles of sulfo-NHS in another 250 microliters of the MES buffer. Combine these solutions with the PEG-modified gold nanoparticles and incubate at 37 degrees Celsius for 30 minutes. Centrifuge the mixture for five minutes and discard the supernatant.
Resuspend the pellet in 500 microliters of the MES buffer. Add to the suspension a sufficient quantity of IgG antibody for an antibody concentration of 100 picograms per milliliter. Incubate the mixture at 37 degrees Celsius for two hours.
Centrifuge the mixture for 10 minutes and remove the supernatant. Resuspend the pellet in 500 microliters of the MES buffer and repeat the centrifugation. Then, resuspend the pellet in 500 microliters of a pH 7.5 buffer of 10 millimolar sodium phosphate and 0.3 molar sodium chloride in deionized water.
Incubate the mixture at 37 degrees Celsius for 30 minutes. Centrifuge the mixture for 10 minutes twice, as previously described. Resuspend the pellet in 500 microliters of 1%BSA in 10 millimolar pH six MES buffer and incubate the mixture at four degrees Celsius overnight.
Centrifuge the mixture for 10 minutes twice. Finally, resuspend the pellet in 500 microliters of MES buffer to obtain the IgG modified gold nanoparticles. Use a robotic printer to deposit microarrays of a protein G concentration gradient on nitrocellulose membranes.
Next, load a microarray into a stainless steel filter holder of an appropriate size. Connect a syringe containing an eight picomolar suspension of IgG modified gold nanoparticles to the inlet of the filter holder. Mount the syringe on a syringe pump.
Set up a 50-milliliter tube to collect the outlet flow from the filter holder. Then, flow the suspension through the filter holder at one milliliter per minute. Afterwards, allow the microarray to dry for 30 minutes at room temperature.
Clean and dry the filter holder with water and filter paper. Scan the dry microarray in a flatbed scanner at 4, 800 DPI and save the image as a 24-bit color TIFF file. Next, prepare both a five millimolar chloroauric acid solution and a 1.027 molar hydrogen peroxide solution in 10 millimolar pH six MES buffer.
To premix the enhancement solution, place equal values of the chloroauric acid and hydrogen peroxide solutions in a microcentrifuge tube and thoroughly mix the solutions. Apply the premixed enhancement solution to the microarray sensor surface. Alternatively, apply equal volumes of the chloroauric acid and hydrogen peroxide solutions to the sensor surface and thoroughly mix them directly on the sensor.
Incubate the sensor with the enhancement solution for up to five minutes, then submerge the microarray in deionized water for five seconds to rinse away the enhancement solution. Allow the microarray to air dry at room temperature. Repeat this process for each microarray to be enhanced.
Scan the enhanced microarrays and save the images. In a standard paper-based immunoassay, IgG modified gold, silver and iron oxide nanoparticles detected protein G concentrations at two times 10 to the fifth, two times 10 to the fourth, and two times 10 to the seventh molecules per spot, respectively. No visual signal was observed from IgG modified silicon nanoparticles.
Incubating the microarrays in enhancement solution resulted in a 100-fold signal increase across the gold, silver and iron oxide nanoparticles. In the case of silicon nanoparticles, this enhancement allowed a visual signal to be observed. This enhancement was applied to a commercial glass-based allergen component microarray immunoassay of four human serum samples.
Staining with anti-IgE labeled gold nanoparticles and enhancing the signal had comparable results to the standard fluorometric assay. The average linear correlation between the mean fluorescence intensity and the mean colorimetric intensity was 0.79. Once learned, this technique can be done in under 10 minutes if performed correctly.
Overview
This article presents a protocol for enhancing signals in nanoprobe-based biosensing by reducing chloroauric acid onto various nanoparticle surfaces. The method allows for the detection of low concentrations of nanoparticles, demonstrating versatility across different substrates.
Key Study Components
Area of Science
- Nanoparticle-based biosensing
- Signal enhancement techniques
- Colorimetric assays
Background
- Nanoprobes are used in various biosensing applications.
- Signal amplification is crucial for detecting low-abundance targets.
- Chloroauric acid reduction enhances the signal of existing nanoprobes.
- This technique can be adapted for different nanoparticle types and substrates.
Purpose of Study
- To develop a protocol for enhancing signals from nanoparticle-based probes.
- To demonstrate the versatility of the method across different materials.
- To achieve detectable signals with minimal nanoparticle quantities.
Methods Used
- Preparation of gold nanoparticles and PEG modification.
- Use of EDC and sulfo-NHS for antibody conjugation.
- Application of chloroauric acid and hydrogen peroxide for signal enhancement.
- Scanning and imaging of microarrays for analysis.
Main Results
- Signal enhancement resulted in a 100-fold increase for gold, silver, and iron oxide nanoparticles.
- Visual signals were achieved for silicon nanoparticles after enhancement.
- The technique was successfully applied to commercial allergen assays.
- Correlation between fluorescence and colorimetric intensity was established.
Conclusions
- The proposed method significantly improves detection capabilities of nanoparticle-based assays.
- It offers a rapid and adaptable approach for various biosensing applications.
- Future applications may extend to other nanoparticle types and assay formats.
Frequently Asked Questions
Executive Industry Relevance
This signal enhancement protocol addresses a critical bottleneck in nanoprobe-based biosensing: the need for high nanoparticle densities to achieve detectable colorimetric signals. By enabling up to 100-fold signal amplification through in situ gold deposition, the method improves detection sensitivity for low-abundance targets without requiring nanoprobe redesign. This capability supports early-stage target validation and assay development by reducing false negatives and increasing confidence in hit identification across diverse biomarker discovery workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of low-abundance biomarkers by amplifying signals from nanoprobes bound to sparse molecular targets.
- Operational Value: Reduces reagent consumption and assay time by allowing detection with fewer nanoparticles per spot.
Screening & Assay Development
- Scientific Value: Supports assay standardization by providing consistent signal enhancement across gold, silver, iron oxide, and silicon nanoprobes on paper and glass substrates.
- Operational Value: Facilitates screening readiness through rapid (<5 min) enhancement compatible with low-end imaging devices like flatbed scanners or digital cameras.
Translational & Preclinical Research
- Scientific Value: Demonstrated applicability to commercial allergen microarrays shows translational continuity from discovery to clinically relevant sample analysis.
- Operational Value: Enables risk-adjusted advancement by correlating colorimetric output with established fluorometric assays (R=0.79), supporting data cross-validation.
Pipeline & Workflow Integration
The method integrates into the discovery continuum by enhancing readouts after nanoprobe-based target engagement, positioning it between primary assay execution and data analysis stages where signal confidence impacts lead identification decisions.
- Discovery Biology: Supports hypothesis testing by amplifying weak signals that might otherwise fall below detection thresholds in target validation screens.
- Screening: Delivers quantitative, reproducible outputs through standardized enhancement that improves spot-to-spot consistency in microarray formats.
- Analytics: Generates colorimetric intensity measurements enabling comparison across conditions and supporting statistical evaluation of binding events.
- Translational Research: Connects discovery to preclinical relevance via successful use in human serum allergen profiling, maintaining biomarker signal integrity.
- Enterprise Reuse: Represents a modular enhancement step applicable across multiple nanoprobe types and assay platforms, reducing need for assay-specific optimization.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target engagement by lowering detection limits to as few as 10 nanoparticles per spot.
- Operational Value: Enhances reproducibility and scalability through buffer-based enhancement compatible with automated microarray processing.
- Strategic Value: Improves capital efficiency by extending utility of existing nanoprobe inventories and reducing repeat testing due to false negatives.
- Portfolio Impact: Enables better go/no-go decisions by increasing signal-to-noise ratio in early screening, reducing late-stage attrition from poorly validated targets.
Implementation Considerations
- Requires expertise in nanoprobe conjugation and microarray handling for consistent surface functionalization.
- Depends on access to chloroauric acid, hydrogen peroxide, and MES buffer solutions with controlled mixing and incubation timing.
- Necessitates standardization of enhancement application (premixed vs. on-surface) across teams to ensure signal uniformity.
- Involves adaptation considerations for different nanoparticle core materials (e.g., silicon vs. metal oxide) to optimize deposition efficiency.
- Practical limitation: Enhancement efficacy depends on nanoprobe surface accessibility and may require optimization for densely packed or sterically hindered assays.
