Site-Directed Electrochemical Grafting for Amplified Detection of Antibody Pharmaceuticals.

Antibody pharmaceuticals have become the most popular immunotherapeutic drugs and are often administered with low serum drug dosages. Hence, the development of a highly sensitive method for the quantitative assay of antibody levels is of great importance to individualized therapy. On the basis of the dual signal amplification by the glycan-initiated site-directed electrochemical grafting of polymer chains (glyGPC), we report herein a novel strategy for the amplified electrochemical detection of antibody pharmaceuticals. The target of interest was affinity captured by a DNA aptamer ligand, and then the glycans of antibody pharmaceuticals were decorated with the alkyl halide initiators (AHIs) via boronate cross-linking, followed by the electrochemical grafting of the ferrocenyl polymer chains from the glycans of antibody pharmaceuticals through the electrochemically controlled atom transfer radical polymerization (eATRP). As the glycans can be decorated with multiple AHIs and the grafted polymer chains are composed of tens to hundreds of electroactive tags, the glyGPC-based strategy permits the dually amplified electrochemical detection of antibody pharmaceuticals. In the presence of trastuzumab (Herceptin) as the target, the glyGPC-based strategy achieved a detection limit of 71.5 pg/mL. Moreover, the developed method is highly selective, and the results of the quantitative assay of trastuzumab levels in human serum are satisfactory. Owing to its uncomplicated operation and cost-effectiveness, the glyGPC-based strategy shows great promise in the amplified electrochemical detection of antibody pharmaceuticals.

Target-Synergized Biologically Mediated RAFT Polymerization for Electrochemical Aptasensing of Femtomolar Thrombin.

The assay of thrombin levels is integral to the assessment of coagulation function and clinical screening of coagulation disorder-related diseases. In this work, we illustrate the ingenious use of the target-synergized biologically mediated reversible addition-fragmentation chain transfer (RAFT) polymerization (tsBMRP) as a novel amplification strategy for the electrochemical aptamer-based biosensing of thrombin at the femtomolar levels. Briefly, the tsBMRP-based strategy relies on the boronate affinity-mediated decoration of the glycan chain(s) of the target itself with RAFT agents and the subsequent recruitment of signal labels via BMRP, mediated by the direct reduction of RAFT agents by NADH into initiating/propagating radicals. Obviously, the tsBMRP-based strategy is biologically friendly, low-cost, and simple in operation. As thrombin is a glycoconjugate, its electrochemical aptasensing involves the use of the thrombin-binding aptamer (TBA) as the recognition receptor, the site-specific decoration of RAFT agents to the glycan chain of thrombin via boronate affinity, and further the recruitment of ferrocene signal labels via the BMRP of ferrocenylmethyl methacrylate (FcMMA). As boronate affinity results in the decoration of each glycan chain with tens of RAFT agents while BMRP recruits hundreds of signal labels to each RAFT agent-decorated site, the tsBMRP-based strategy allows us to detect thrombin at a concentration of 35.3 fM. This electrochemical aptasensor is highly selective, and its applicability to thrombin detection in serum samples has been further demonstrated. The merits of high sensitivity and selectivity, low cost, good anti-interference capability, and simple operation make the tsBMRP-based electrochemical thrombin aptasensor great promise in biomedical and clinical applications.

Amplification-Free Ratiometric Electrochemical Aptasensor for Point-of-Care Detection of Therapeutic Monoclonal Antibodies.

The rapid quantification of therapeutic monoclonal antibodies (mAbs) is of great significance to their pharmacokinetics/pharmacodynamics (PK/PD) research and the personalized medication for disease treatment. Taking advantage of the direct decoration of tens of redox tags to the target of interest, we illustrate herein an amplification-free ratiometric electrochemical aptasensor for the point-of-care (POC) detection of trace amounts of therapeutic mAbs. The POC detection of therapeutic mAbs involved the use of the methylene blue (MB)-conjugated aptamer as the affinity element and the decoration of therapeutic mAbs with ferrocene (Fc) tags via the boronate crosslinking, in which the MB-derived peak current was used as the reference signal, and the peak current of the Fc tag was used as the output signal. As each therapeutic mAb carries tens of diol sites for the site-specific decoration of the Fc output tags, the boronate crosslinking enabled the amplification-free detection, which is cost-effective and quite simple in operation. In the presence of bevacizumab (BevMab) as the target, the resulting ratiometric signal (i.e., the IFc/IMB value) exhibited a good linear response over the range of 0.025-2.5 µg/mL, and the limit of detection (LOD) of the electrochemical aptasensor was 6.5 ng/mL. Results indicated that the aptamer-based affinity recognition endowed the detection of therapeutic mAbs with high selectivity, while the ratiometric readout exhibited satisfactory reproducibility and robustness. Moreover, the ratiometric electrochemical aptasensor is applicable to the detection of therapeutic mAbs in serum samples. Taking together, the amplification-free ratiometric electrochemical aptasensor holds great promise in the POC detection of therapeutic mAbs.

Dually Amplified Electrochemical Aptasensor for Endotoxin Detection via Target-Assisted Electrochemically Mediated ATRP.

As the entering of bacterial endotoxin into blood can cause various life-threatening pathological conditions, the screening and detection of low-abundance endotoxin are of great importance to human health. Taking advantage of signal amplification by target-assisted electrochemically mediated atom transfer radical polymerization (teATRP), we illustrate herein a simple and cost-effective electrochemical aptasensor capable of detecting endotoxin with high sensitivity and selectivity. Specifically, the aptamer receptor was employed for the selective capture of endotoxin, of which the glycan chain was then decorated with ATRP initiators via covalent coupling between the diol sites and phenylboronic acid (PBA) group, followed by the recruitment of ferrocene signal reporters via the grafting of polymer chains through potentiostatic eATRP under ambient temperature. As the glycan chain of endotoxin can be decorated with hundreds of ATRP initiators while the further grafting of polymer chains through eATRP can recruit hundreds to thousands of signal reporters to each initiator-decorated site, the teATRP-based strategy allows for the dual amplification of the detection signal. This dually amplified electrochemical aptasensor has the ability to sensitively and selectively detect endotoxin at a concentration as low as 1.2 fg/mL, and its practical applicability has been further demonstrated using human serum samples. Owing to the simplicity, high efficiency, biocompatibility, and inexpensiveness of the teATRP-based amplification strategy, this electrochemical aptasensor holds great application potential in the sensitive and selective detection of low-abundance endotoxin and many other glycan chain-containing bio-targets.

Boronate Affinity-Amplified Electrochemical Aptasensing of Lipopolysaccharide.

As lipopolysaccharide (LPS) is closely associated with sepsis and other life-threatening conditions, the point-of-care (POC) detection of LPS is of significant importance to human health. In this work, we illustrate an electrochemical aptasensor for the POC detection of low-abundance LPS by utilizing boronate affinity (BA) as a simple, efficient, and cost-effective amplification strategy. Briefly, the BA-amplified electrochemical aptasensing of LPS involves the tethering of the aptamer receptors and the BA-mediated direct decoration of LPS with redox signal tags. As the polysaccharide chain of LPS contains hundreds of cis-diol sites, the covalent crosslinking between the phenylboronic acid group and cis-diol sites can be harnessed for the site-specific decoration of each LPS with hundreds of redox signal tags, thereby enabling amplified detection. As it involves only a single-step operation (∼15 min), the BA-mediated signal amplification holds the significant advantages of unrivaled simplicity, rapidness, and cost-effectiveness over the conventional nanomaterial- and enzyme-based strategies. The BA-amplified electrochemical aptasensor has been successfully applied to specifically detect LPS within 45 min, with a detection limit of 0.34 pg/mL. Moreover, the clinical utility has been validated based on LPS detection in complex serum samples. As a proof of concept, a portable device has been developed to showcase the potential applicability of the BA-amplified electrochemical LPS aptasensor in the POC testing. In view of its simplicity, rapidness, and cost-effectiveness, the BA-amplified electrochemical LPS aptasensor holds broad application prospects in the POC testing.

Electrochemical Detection of Femtomolar DNA via Boronate Affinity-Mediated Decoration of Polysaccharides with Electroactive Tags.

In view of their high efficiency and cost-effectiveness, polymers are of great promise as carriers for signal tags in amplified detection. Herein, we present a polysaccharide-amplified method for the electrochemical detection of a BRCA1 breast cancer gene-derived DNA target at the femtomolar levels. Briefly, peptide nucleic acid (PNA) with a complementary sequence was tethered as the capture probe for the DNA target, to which carboxyl group-containing polysaccharides were then attached via facile phosphate-Zr(IV)-carboxylate crosslinking, followed by the decoration of polysaccharide chains with electroactive ferrocene (Fc) signal tags via affinity coupling between a cis-diol site and phenylboronic acid (PBA) group. As the polysaccharide chain contains hundreds of cis-diol sites, boronate affinity can enable the site-specific decoration of each polysaccharide chain with hundreds of Fc signal tags, efficiently transducing each target capture event into the decoration of many Fc signal tags. As polysaccharides are cheap, renewable, ubiquitous, and biodegradable natural biopolymers, the use of polysaccharides for signal amplification offers the benefits of high efficiency, cost-effectiveness, excellent biocompatibility, and environmental friendliness. The linear range of the polysaccharide-amplified method for DNA detection was demonstrated to be from 10 fM to 10 nM (R2 = 0.996), with the detection limit as low as 2.9 fM. The results show that this method can also discriminate single base mismatch with satisfactory selectivity and can be applied to DNA detection in serum samples. In view of these merits, the polysaccharide-amplified PNA-based electrochemical method holds great promise in DNA detection with satisfactory sensitivity and selectivity.

Boronate-Affinity Cross-Linking-Based Ratiometric Electrochemical Detection of Glycoconjugates.

Despite the widespread application of the boronate-affinity cross-linking (BAC) in the separation, enrichment, and sensing of glycoconjugates, it remains a huge challenge to integrate the BAC into the selective electrochemical detection of glycoconjugates due to the poor selectivity of the BAC. Herein, we demonstrate a BAC-based ratiometric electrochemical method for the simple, low-cost, and highly sensitive and selective detection of glycoconjugates. Briefly, the methylene blue (MB)-tagged nucleic acid aptamer is exploited as the recognition element to selectively capture target glycoconjugate, to which a large number of ferrocene (Fc) tags are subsequently labeled via the BAC between the phenylboronic acid (PBA) group and the cis-diol site of the oligosaccharide chains on the captured targets. Using the MB tag as the internal reference and the Fc tag as the reporter of the target capture, the dual-signal output enables the ratiometric detection. Due to the presence of a high density of the cis-diol sites on a glycoconjugate, sufficiently high sensitivity can be obtained even without using any amplification strategies. Using glycoprotein mucin 1 (MUC1) as the model target, the signal ratio (IFc/IMB) exhibits good linearity over the range from 0.05 to 50 U/mL, with a detection limit of 0.021 U/mL. In addition to the high sensitivity and selectivity, the results of the analysis of MUC1 in serum samples are acceptable. By virtue of its simplicity, cost-effectiveness, and high robustness and reproducibility, this BAC-based ratiometric electrochemical method holds great promise in the highly sensitive and selective detection of glycoconjugates.

Boronate Affinity-Based Electrochemical Aptasensor for Point-of-Care Glycoprotein Detection.

As a class of oligosaccharide chain-containing proteins, glycoproteins are of great value in screening and early diagnosis of malignant tumors and other major diseases. Herein, we report a universal boronate affinity-based electrochemical aptasensor for point-of-care glycoprotein detection. Aptasensing of glycoproteins involves the specific recognition and capture of target glycoproteins by end-tethered nucleic acid aptamers and the site-specific labeling of ferrocene tags via the phenylboronic acid (PBA)-based boronate affinity interactions because the cis-diol sites of oligosaccharide chains on glycoproteins can selectively react with the PBA receptor groups to form cyclic phenylborates in aqueous basic media. Due to the presence of hundreds to thousands of cis-diol sites on a glycoprotein, a large number of ferrocene tags can be recruited for the signal-on aptasensing of glycoproteins at a low-abundance level, eliminating the need for extra amplification strategies. As a result, the boronate affinity-based electrochemical aptasensor is highly sensitive and selective for glycoprotein detection and tolerant to the false-positive results. The detection limit for α-fetoprotein (AFP) is 0.037 ng/mL, with a linear response ranging from 0.1 to 100 ng/mL. In addition to the merits of simple operation, short assay time, and low detection cost, the aptasensor is applicable to the detection of glycoproteins in serum samples and the point-of-care detection using disposable flexible electrodes. Overall, this work provides a universal and promising platform for the point-of-care detection of glycoproteins, holding great potential in screening and early diagnosis of glycoprotein-related malignant tumors and other major diseases.

Target-mediated silver deposition-based electrochemical biosensor for highly sensitive detection of human chorionic gonadotropin.

As a glycoprotein hormone, human chorionic gonadotropin (hCG) is an established marker for pregnancy test. On the basis of the target-mediated silver deposition (TSD), in this work, we report the development of an amplification-free electrochemical biosensor for the highly sensitive detection of hCG. The detection of hCG involves the use of the affinity peptide-modified electrode for hCG capture (the CGGSSPPLRINRHILTR peptide containing the hCG-binding domain of the PPLRINRHILTR sequence is used as the affinity peptide), the oxidation of the diol sites of the glycan chains on hCG hormones into aldehyde groups by NaIO4, and the deposition of silver nanoparticles (AgNPs) for the solid-state voltammetric stripping analysis. Due to the deposition of multiple AgNPs while the solid-state Ag/AgCl voltammetric process has a high signal-to-noise ratio, the TSD-based electrochemical biosensor can be applied to the highly sensitive detection of hCG without the need for signal amplification. Under optimal conditions, the stripping current increased linearly with an increasing hCG concentration over the range from 1.0 to 25 mIU/mL, with a detection limit of 0.45 mIU/mL. Owing to the high specificity of the hCG-binding peptide PPLRINRHILTR, this electrochemical hCG biosensor exhibits high selectivity. The results of the quantitative assay of hCG in urine samples at the concentrations of 25, 10, and 1.0 mIU/mL are desirable, indicating the good anti-interference capability. As the TSD-based electrochemical biosensor allows the amplification-free detection of low-abundance hCG, it is easy to use and cost-effective, showing great promise in point-of-care assay of hCG for pregnancy test.

Boronate crosslinking-based ratiometric electrochemical assay of glycated albumin.

As a product of nonenzymatic glycation, glycated albumin (GA) is a promising serum marker for the short-term glycemic monitoring in patients with diabetes. On the basis of the boronate crosslinking (BCL)-enabled direct labeling of ferrocene (Fc) tags to the nonenzymatically glycated (NEG) sites, we report herein a novel aptamer-based ratiometric electrochemical (apt-REC) platform for the point-of-care (POC) assay of GA. This apt-REC platform is based on the recognition of GA proteins by the methylene blue (MB)-modified aptamer receptors and the labeling of the Fc tags to the NEG sites via the BCL. Using MB as the reference tag and Fc as the quantification tag, the ratio of the oxidation currents (i.e., IFc/IMB) can serve as the yardstick for the ratiometric assay of GA. Due to the presence of tens of the NEG sites, each GA protein can be labeled with tens of quantification tags, permitting the amplified assay in a simple, time-saving, and low-cost manner. The ratiometric signal exhibited a good linear response over the range from 0.1 to 100 µg/mL, with a detection limit of 45.5 ng/mL. In addition to the superior reproducibility and robustness, this apt-REC platform is highly selective (capable of discriminating GA against human serum albumin (HSA)) and applicable to GA assay in serum samples. Due to its low cost, high reproducibility and robustness, simple operation, and high sensitivity and selectivity, this apt-REC platform holds great promise in the POC assay of GA for diabetes management.