Toward Liquid Biopsy: Determination of the Humoral Immune Response in Cancer Patients Using HaloTag Fusion Protein-Modified Electrochemical Bioplatforms

Abstract

Autoantibodies raised against tumor-associated antigens have shown high promise as clinical biomarkers for reliable diagnosis, prognosis, and therapy monitoring of cancer. An electrochemical disposable biosensor for the specific and sensitive determination of p53-specific autoantibodies has been developed for the first time in this work. This biosensor involves the use of magnetic microcarriers (MBs) modified with covalently immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric signal using the system hydroquinone/HO was related to the levels of p53-autoantibodies in the sample. The biosensor was applied for the analysis of sera from 24 patients with high-risk of developing colorectal cancer and 6 from patients already diagnosed with colorectal (4) and ovarian (2) cancer. The developed biosensor was able to determine p53 autoantibodies with a sensitivity higher than that of a commercial standard ELISA using a just-in-time produced protein in a simpler protocol with less sample volume and easily miniaturized and cost-effective instrumentation.

Electrochemical Biosensors for Determination of Colorectal Tumor Biomarkers

“…Recently, a novel multiplexed electrochemical biosensor for the simultaneous detection of CRC-specific autoantibodies in plasma samples was developed by Garranzo-Asensio et al [48], similarly to that previously reported for a single tumor-associated antigen (TAA) (p53) [131]. The multiplexed immunosensor used HaloTag fusion proteins self-assembled on magnetic microparticles and amperometric detection at disposable SPCEs in the presence of the H 2 O 2 /HQ system ( Figure 17).…”

“…Therefore, the level of serum p53 antibodies is a potential biomarker of high stability for minimally invasive cancer malignancy screening, monitoring, and prognosis [130]. Garranzo-Asensio et al [131] developed a biosensor based on magnetic beads (MBs) modified with covalently immobilized HaloTag fusion p53 protein as an electrochemical detection system ( Figure 6). After capturing the magnetic beads bearing the immunocomplexes onto screen-printed carbon working electrodes, the bio-recognition event was monitored by the amperometric readout generated by the enzymatic reduction of H 2 O 2 mediated by HQ, which revealed the level of p53 autoantibodies in the sample.…”

“…Schematic illustration of the magnetic beads (MBs) immunosensor based on the HaloTagmodified fusion protein for the detection of p53-specific autoantibodies. Reprinted from[131] with permission. Copyright © 2016, American Chemical Society.…”

Ultrarapid Measurement of Diagnostic Antibodies by Magnetic Capture of Immune Complexes

“…POCT is aimed at bringing immediate test results to the patient, physician, and care team, thereby increasing the likelihood that immediate clinical management decisions can be made. Although many immunoassays have been described for detecting clinically relevant antibodies, including ELISAs and new technologies employing nanoplasmonic biosensors or electrochemical detection 4–6 , these methods are impractical for POCT due to prolonged assay times and complicated procedures. Currently, solid-phase lateral flow immunoassays and miniaturized ELISAs have been developed for POCT.…”

Improving Cancer Outcomes through Electrochemical Biosensing of Early Diagnosis/Prognosis Biomarkers in Human Biopsies

“…Within this context, the main characteristics of novel biosensing scaffolds for the determination of cancer related-miRNAs [1][2][3] and autoantibodies [4] against tumor associated antigens (TAAs), relevant biomarkers considered for both early diagnosis and prognosis, will be presented. The developed methodologies, based on the coupling of attractive bioreceptors and bioassay formats, functionalized magnetic microcarriers, and electrochemical detection at disposable transducers, have demonstrated practical applicability for the accurate determination of the endogenous concentration of the target analytes in solid (fresh and FFPE breast human tissues) and liquid (human serum from colorectal and ovarian cancer patients) biopsies.…”