Highly Reusable Electrochemical Immunosensor for Ultrasensitive Protein Detection

Abstract

The detection and quantification of protein biomarkers in bodily fluids is important for many clinical applications, including disease diagnosis and health monitoring. Current techniques for ultrasensitive protein detection, such as enzyme-linked immunosorbent assay (ELISA) and electrochemical sensing, involve long incubation times (1.5-3 hr) and rely on single-use sensing electrodes which can be costly and generate excessive waste. This work demonstrates a reusable electrochemical immunosensor employing magnetic nanoparticles (MNPs) and dually labeled gold nanoparticles (AuNPs) for ultrasensitive measurements of protein biomarkers. As proof of concept, this platform was used to detect C-X-C motif chemokine ligand 9 (CXCL9), a biomarker associated with kidney transplant rejection, immune nephritis from checkpoint inhibitor therapy, and drug-associated acute interstitial nephritis, in human urine. The sensor successfully detected CXCL9 at concentrations as low as 27 pg/mL within ~1 hr. This immunosensor was also adapted onto a handheld smartphone-based diagnostic device and used for measurements of CXCL9, which exhibited a lower limit of detection of 65 pg/mL. Lastly, we demonstrate that the sensing electrodes can be reused for at least 100 measurements with a negligible loss in analytical performance, reducing the costs and waste associated with electrochemical sensing.

Keywords: CXCL9; diagnostic; electrochemical; gold nanoparticles; immunosensor; magnetic nanoparticles; reusable.

Figure 1.. Workflow of the Reusable Electrochemical Immunosensor.

(A) The sample is incubated with cAb-MNPs and dually labeled AuNPs, resulting in the formation of MNP-antigen-AuNP immunocomplexes. (B) MNP-antigen-AuNP immunocomplexes are separated from the sample using a magnetic rack. (C) TMB substrate is added to the MNP-antigen-AuNP immunocomplexes and the solution is dispensed onto the SPGE sensor. The sensor is placed on a magnetic stage resulting in the MNP-antigen-AuNP immunocomplexes being concentrated onto the working electrode (i). A bias potential is applied to the reference and working electrodes, and an electrochemical current is generated by the HRP-TMB substrate redox reaction (ii). (D) The electrochemical signal is read using a smartphone-based diagnostic device or a benchtop potentiostat. (E) The sensor is gently rinsed with wash buffer and dried using compressed air, enabling it to be reused.

Figure 2.. Optimization of Electrochemical Immunoassay Parameters.

(A) SBRs obtained from human urine samples spiked with CXCL9 at 0 pg/mL or 10,000 pg/mL using AuNPs labeled with various dAb:HRP molar ratios. Nanoparticles were incubated with the sample for 30 min. (B) SBRs obtained from CXCL9-spiked and nonspiked urine samples using varying amounts of cAb-MNPs. (C) SBRs obtained from CXCL9-spiked and nonspiked urine samples using varying AuNP-to-MNP ratios. Measurements were performed using 5 μL of MNPs. (D) SBRs obtained from CXCL9-spiked and nonspiked urine samples with a 30 min or 60 min sample incubation period. (E) SBRs obtained from CXCL9-spiked and nonspiked urine samples with varying streptavidin (SA)-HRP incubation times. (F) SBRs obtained from CXCL9-spiked and nonspiked urine samples with varying times for magnetic attraction. Each bar represents the mean ± standard deviation (SD) of three independent measurements. Statistical significance was determined using a one-way ANOVA for panels A-C and E-F, and using a student’s T-test for panel D. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

Figure 3.. Analytical Performance of the Reusable Electrochemical Immunosensor.

(A) Chronoamperograms generated from urine spiked with CXCL9 at concentrations from 0 to 10,000 pg/mL with a 60 min incubation. Each line represents the average current produced using 3 independent measurements at each concentration. (B) Calibration plots based on amperometric currents at 150 sec obtained from chronoamperograms in panel A. Measurements were carried out using a benchtop potentiostat. Each point represents the mean ± SD of 3 independent measurements. (C) Amperometric currents generated by the electrochemical immunosensor for measurements of urine samples spiked with CXCL9 from 0 pg/mL to 100,000 pg/mL using a benchtop potentiostat and smartphone-based diagnostic device. Each bar represents the mean ± SD of three individual measurements. (D) Comparison of CXCL9 concentrations in spiked urine samples determined by the electrochemical immunosensor and a commercial ELISA kit. Each data point represents the mean ± SD of 3 replicate measurements at each concentration.

Figure 4.. Reusability of the Electrochemical Immunosensor.

(A) Amperometric currents produced by three separate sensing electrodes at 0 pg/mL and 10,000 pg/mL of CXCL9 spiked in urine. Each bar represents the mean ± SD of 50 measurements. (B) SBRs obtained from urine samples spiked with CXCL9 at 0 pg/mL or 10,000 pg/mL using fresh sensing electrodes and sensing electrodes reused for 100 measurements. Each bar represents the mean ± SD of 3 measurements obtained using 3 distinct sensors. Statistical significance was assessed using a student’s t test with significance defined as p<0.05. (C) SBRs obtained from urine samples spiked with CXCL9 at 0 pg/mL or 10,000 pg/mL using the same sensing electrodes over the course of 14 days. Each bar represent the mean ± SD of 3 measurements. Statistical significance was assessed using a one-way ANOVA with significance defined as p<0.05.