A comparative study of bioconjugation solutions for enhanced sensitivity in gold nanoparticle-based assays

Abstract

Gold nanoparticles (AuNPs) incorporated into lateral flow assays (LFAs) provide a low-cost, simple and rapid pointof-care solution for diagnosing a broad spectrum of diseases. Kimialys’ patented K-One surface chemistry controls the conjugation of antibodies at the AuNPs surface to provide rapid and efficient bioconjugation.

This white paper assesses the performance of the Kimialys’ bioconjugation kit against three commercially available competitor kits. In collaboration with our partners from CEA Paris-Saclay, we immobilized anti-ricin antibodies to AuNPs using the four bioconjugation kits and manufactured dipstick assays. Stable bioconjugates were produced by all four kits, leading to reproducible detection.

After optimization of the four bioconjugation kits, Kimialys’ protocol delivered the highest bioconjugation yield in the shortest time (90.5% in 35 mins) and the most sensitive visual detection with a 4-fold gain in visual LoD compared with the performance of the closest competitor kit (Kimialys, LoD:12 pg/mL; Competitor 1, LoD: 50 pg/mL). Automated optical LoD assessment confirmed that Kimialys’ kit produced the most sensitive LFA. The estimated analytical LoD was 2.1 pg/mL, an 11-fold theoretical gain in sensitivity compared with the closest competitor kit.

These data demonstrate the Kimialys bioconjugation kit leads to a more efficient bioconjugate production (higher yield and shorter protocol) and more sensitive detection (improved LoD) in LFAs, which is key for clinical applications.

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Introduction

Lateral flow assays (LFAs) allow the detection and quantification of analytes from complex patient sample solutions. LFAs offer low-cost options that do not need specialist staff or laboratory capacity (1, 2) for self-testing or regular testing at low-infrastructure testing sites (3, 4). Effective LFAs are rapid and allow the sensitive detection of a biomarker or analyte. It is crucial to keep LFA component costs low by increasing the efficiency of the bioconjugation reaction whilst achieving sensitive detection.

Kimialys has finetuned its surface chemistry technology to control the orientation, surface density and distribution of bioreceptors on the nanoparticle. Background noise is also reduced by protecting the entire particle surface from non-specific interactions. Kimialys strives to keep costs down by optimizing its bioconjugation chemistry whilst producing highly sensitive detection assays.

This study used ricin as the analyte as there is a growing demand for reliable and economical tests to detect ricin due to potential bioterrorism attacks (5, 6). Here we compare the performance of Kimialys’ bioconjugation technology with three competitor kits. We analyze the efficiency of the bioconjugation reaction and monitor the stability of the bioconjugates. We assess the performance of the resulting LFAs in a dipstick assay format using visual and automated LoD. These data show that Kimialys’ efficient bioconjugation chemistry produces more sensitive LFAs than existing kits.

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Methods

Dipstick assay

The performance of the bioconjugates was assessed in a dipstick rapid test format applied to the detection of ricin toxin. The anti-ricin antibody AuNPs bioconjugates were mixed with the ricin sample and applied to the dipstick. The dipsticks provided by CEA Paris-Saclay were ready-to-use with sprayed control and test lines. The strips comprised a sample pad (glass fibre) and an absorbent pad. The concentrations of detected ricin, diluted in a running buffer provided by CEA Paris-Saclay, ranged from 5000 pg/mL to 5 pg/mL in a final sample volume of 100 μL. The bioconjugates (10 μL) were used at an optical density (OD) 8, and the assays were left to migrate for 30 mins before detection. Both the test (T) and control (C) lines of the dipstick strips were analyzed. The LoD was determined as the lowest ricin concentration reliably detected visually by two users trained to interpret lateral flow assays or the automated test reader (iPeak® reader).

Except for the bioconjugation kits, all the materials and reagents needed to perform rapid detection of ricin were provided by our partners from the Immunoanalysis Studies and Research Laboratory (CEA Paris-Saclay), a leading French research and innovation centre with a strong experience in lateral flow assay development. CEA Paris-Saclay manufactured the research use only (RUO) lateral flow strips used in this study.

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Characterization of bioconjugates

UV-Vis spectrophotometry was used to assess the stability of the bioconjugates by scanning the spectra ofbioconjugates in the UV-Visible region. Batch-to-batch reproducibility was evaluated by analyzing the UV-Visible spectra of three batches of bioconjugates from each kit.

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Conjugation kits

All the kits used in this study consisted of 40 nm AuNPs with covalently immobilized antibodies. The study included the following conjugation kits: Kimialys kit (40 nm AuNP + K-One chemistry), Competitor 1 (freezedried Gold Conjugation Kit 40 nm OD20), Competitor 2 lyophilized NHS 40 nm Gold) and competitor 3 (40 nm NHS activated Gold conjugation). The three competitor kits were supplied as pre-functionalized, preactivated lyophilized AuNPs. The Kimialys kit used AuNPs prefunctionalized with K-One surface chemistry in solution, ready to be activated and bioconjugated.

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Results

Bioconjugation using manufacturers’ optimized protocols

Each kit was initially conjugated using the manufacturers’ optimization instructions: Kimialys, competitor kits 1 and 2 used 5 μg of anti-ricin antibody for 1 mL OD=1 of AuNPs. For competitor kit 3, 40 μg of anti-ricin antibody were used for 1 mL OD=2 of AuNPs.

First, the efficiency of the bioconjugation reaction was assessed by analyzing the bioconjugate stability (data not shown), reaction time and bioconjugation yield (Table 1). The Kimialys bioconjugation kit was the quickest, with the highest conjugation yield at 90.5% compared with the three competitor kits.

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Table 1. Summary table of the bioconjugation efficiencies using the instructions for use

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Next, the performance of the bioconjugation kits was assessed in LFAs in a dipstick format by determining the visual LoD. The Kimialys kit exhibited the highest sensitivity of all the conjugation kits, with a visual LoD of 12 pg/ml of ricin (Figure 1). The three competitor kits failed to detect the test line at 50 pg/ml (Figure 1).

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Optimization of competitor kits using Kimialys bioconjugation protocol

Different conditions were tested to improve the assay sensitivity reached with the three competitor kits. Increasing the antibody concentration or incubation time used for the bioconjugation reaction did not improve the assay sensitivity. However, exchanging the recommended bioconjugation conditions for the Kimialys’ bioconjugation protocol improved the visual LoD to 50 pg/mL for competitor kit 1 (Figure 2) whilst improving its yield from 87.5% to 92.5% and reducing the time taken for the complete protocol by 2 hrs.

Using the Kimialys buffer conditions instead of the original buffer also improved the bioconjugation yield of competitor kit 2 from 80% to 96% and improved the visual LoD to 125 pg/mL (Figure 2).

The Kimialys protocol failed to produce bioconjugated AuNPs with competitor kit 3, and optimization of this kit using other conditions was not possible.

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Kimialys’ bioconjugates produced the most sensitive LFA

The performance of the dipstick assays was further confirmed and quantified using an automated test reader(iPeak® reader). The iPeak reader measured the intensity of each test and control line. Automated LoD assessment found the LoD of the Kimialys kit (5 pg/mL) was 5-fold more sensitive than the closest competitor assay (competitor kit 1, LoD: 25 pg/mL) (Table 2). Automated detection of the test line confirmed that the Kimialys kit was the most effective dipstick assay. For all ricin concentrations, the Kimialys kit produced the test lines with the highest signal intensities (Figure 3a) and the most intense control line with the least variation in signal intensity (Figure 3b).

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Table 2. Automated LoD detection confirms that the Kimialys kit produces the most sensitive LFA.

The automated test reader (iPeak reader) measured the intensity of the test and control lines for each dipstick assay. The detectable signal intensity threshold for a positive test with the iPeak reader is 20 a.u.

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The analytical LoD was defined by the concentration of detected ricin at which the reader intensity was 20 a.u. This resulted in an 11-fold theoretical gain (Figure 3a and Table 3) in sensitivity for the Kimialys bioconjugates compared with the next most efficient kit (Competitor 1).

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Table 3. The Kimialys kit has the highest estimated analytical limit of detection compared with competitor kits.

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The LoD corresponded to the minimum ricin concentration for which the iPeak reader intensity was greater than 20a.u. The analytical LoD corresponded to the ricin concentration measured at the intersection between the linear regression curve and the 20 a.u. threshold of the iPeak reader intensity.