Electrochemical protein biosensors for disease marker detection: progress and opportunities.

The development of artificial intelligence-enabled medical health care has created both opportunities and challenges for next-generation biosensor technology. Proteins are extensively used as biological macromolecular markers in disease diagnosis and the analysis of therapeutic effects. Electrochemical protein biosensors have achieved desirable specificity by using the specific antibody-antigen binding principle in immunology. However, the active centers of protein biomarkers are surrounded by a peptide matrix, which hinders charge transfer and results in insufficient sensor sensitivity. Therefore, electrode-modified materials and transducer devices have been designed to increase the sensitivity and improve the practical application prospects of electrochemical protein sensors. In this review, we summarize recent reports of electrochemical biosensors for protein biomarker detection. We highlight the latest research on electrochemical protein biosensors for the detection of cancer, viral infectious diseases, inflammation, and other diseases. The corresponding sensitive materials, transducer structures, and detection principles associated with such biosensors are also addressed generally. Finally, we present an outlook on the use of electrochemical protein biosensors for disease marker detection for the next few years.

A miniprotein receptor electrochemical biosensor chip based on quantum dots.

Recently protein binders have emerged as a promising substitute for antibodies due to their high specificity and low cost. Herein, we demonstrate an electrochemical biosensor chip through the electronic labelling strategy using lead sulfide (PbS) colloidal quantum dots (CQDs) and the unnatural SARS-CoV-2 spike miniprotein receptor LCB. The unnatural receptor can be utilized as a molecular probe for the construction of CQD-based electrochemical biosensor chips, through which the specific binding of LCB and the spike protein is transduced to sensor electrical signals. The biosensor exhibits a good linear response in the concentration range of 10 pg mL-1 to 1 µg mL-1 (13.94 fM to 1.394 nM) with the limit of detection (LOD) being 3.31 pg mL-1 (4.607 fM for the three-electrode system) and 9.58 fg mL-1 (0.013 fM for the HEMT device). Due to the high sensitivity of the electrochemical biosensor, it was also used to study the binding kinetics between the unnatural receptor LCB and spike protein, which has achieved comparable results as those obtained with commercial equipment. To the best of our knowledge, this is the first example of using a computationally designed miniprotein receptor based on electrochemical methods, and it is the first kinetic assay performed with an electrochemical assay alone. The miniprotein receptor electrochemical biosensor based on QDs is desirable for fabricating high-throughput, large-area, wafer-scale biochips.

Flexible Amperometric Immunosensor Based on Colloidal Quantum Dots for Detecting the Myeloperoxidase (MPO) Systemic Inflammation Biomarker.

Myeloperoxidase (MPO) has been demonstrated to be a biomarker of neutrophilic inflammation in various diseases. Rapid detection and quantitative analysis of MPO are of great significance for human health. Herein, an MPO protein flexible amperometric immunosensor based on a colloidal quantum dot (CQD)-modified electrode was demonstrated. The remarkable surface activity of CQDs allows them to bind directly and stably to the surface of proteins and to convert antigen-antibody specific binding reactions into significant currents. The flexible amperometric immunosensor provides quantitative analysis of MPO protein with an ultra-low limit of detection (LOD) (31.6 fg mL-1), as well as good reproducibility and stability. The detection method is expected to be applied in clinical examination, POCT (bedside test), community physical examination, home self-examination and other practical scenarios.

B-Cell-Epitope-Based Fluorescent Quantum Dot Biosensors for SARS-CoV-2 Enable Highly Sensitive COVID-19 Antibody Detection.

A new antibody diagnostic assay with more rapid and robust properties is demanded to quantitatively evaluate anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity in a large population. Here, we developed a nanometer-scale fluorescent biosensor system consisting of CdSe-ZnS quantum dots (QDs) coupled with the highly sensitive B-cell epitopes of SARS-CoV-2 that could remarkably identify the corresponding antibody with a detection limit of 100 pM. Intriguingly, we found that fluorescence quenching of QDs was stimulated more obviously when coupled with peptides than the corresponding proteins, indicating that the energy transfer between QDs and peptides was more effective. Compared to the traditional enzyme-linked immunosorbent assay (ELISA), the B-cell-epitope-based QD-biosensor could robustly distinguish coronavirus disease 2019 (COVID-19) antibody-positive patients from uninfected individuals with a higher sensitivity (92.3-98.1% positive rates by QD-biosensor vs. 78.3-83.1% positive rates by ELISAs in 207 COVID-19 patients' sera) in a more rapid (5 min) and labor-saving manner. Taken together, the 'QD-peptides' biosensor provided a novel real-time, quantitative, and high-throughput method for clinical diagnosis and home-use tests.

All-solid-state SARS-CoV-2 protein biosensor employing colloidal quantum dots-modified electrode.

Rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody can provide immunological evidence in addition to nucleic acid test for the early diagnosis and on-site screening of coronavirus disease 2019 (COVID-19). All-solid-state biosensor capable of rapid, quantitative SARS-CoV-2 antibody testing is still lacking. Herein, we propose an electronic labelling strategy of protein molecules and demonstrate SARS-CoV-2 protein biosensor employing colloidal quantum dots (CQDs)-modified electrode. The feature current peak corresponding to the specific binding reaction of SARS-CoV-2 antigen and antibody proteins was observed for the first time. The unique charging and discharging effect depending on the alternating voltage applied was ascribed to the quantum confinement, Coulomb blockade and quantum tunneling effects of quantum dots. CQDs-modified electrode could recognize the specific binding reaction between antigen and antibody and then transduce it into significant electrical current. In the case of serum specimens from COVID-19 patient samples, the all-solid-state protein biosensor provides quantitative analysis of SARS-CoV-2 antibody with correlation coefficient of 93.8% compared to enzyme-linked immunosorbent assay (ELISA) results. It discriminates patient and normal samples with accuracy of about 90%. The results could be read within 1 min by handheld testing system prototype. The sensitive and specific protein biosensor combines the advantages of rapidity, accuracy, and convenience, facilitating the implement of low-cost, high-throughput immunological diagnostic technique for clinical lab, point-of-care testing (POCT) as well as home-use test.