Application of bioconjugation chemistry on biosensor fabrication for detection of TAR-DNA binding protein 43

Abstract

A simple-prepare, single-use and cost-effective, in vitro biosensor for the detection of TAR DNA-binding protein 43 (TDP-43), a biomarker of neuro-degenerative disorders, was designed, manufactured and tested. This study reports the first biosensor application for the detection of TDP-43 using a novel biosensor fabrication methodology. Bioconjugation mechanism was applied by conjugating anti-TDP 43 with N-succinimidyl S-acetylthioacetate (SATA) producing a thiol-linked anti-TDP 43, which was used to directly link with gold electrode surface, minimizing the preparation steps for biosensor fabrication and simplifying the biosensor surface. The effectiveness of this bioconjugation mechanism was evaluated and confirmed by FqRRM12 protein, using nuclear magnetic resonance (NMR). The surface coverage of the electrode was analyzed by Time-of-Flight-Secondary Ion Mass Spectrometry (TOF-SIMS). Differential pulse voltammetry (DPV) was acted as the detection transduction mechanism with the use of [Fe(CN)6]3−/4-redox probe. Human TDP-43 peptide of 0.0005 μg/mL to 2 μg/mL in undiluted human serum was analyzed using this TDP-43 biosensor. Interference study of the TDP-43 biosensor using β-amyloid 42 protein and T-tau protein confirmed the specifity this TDP-43 biosensor. This bioconjugation chemistry based approach for biosensor fabrication circumvents tedious gold surface modification and functionalization while enabling specific detection of TDP-43 in less than 1 hr with a low fabrication cost of a single biosensor less than $3.

Recent Advances on Electrochemical Biosensing Strategies toward Universal Point‐of‐Care Systems

“…[54] Thesensing element was asingle-use gold sensor prototype. [55] Ther ecognition element was target specific antibody.T he transduction element was [Ru(NH 3 ) 6 ] 2+/3+ or [Fe(CN) 6 ] 3À/4À based redox system. Unlike conventional use of redox system as surface impedance indicator, [56] this biosensing strategy applied charged redox system for electrostatically interacting with correspondingly charged protein and further quantification by the electrochemical redox reaction based on the reaction kinetic difference.After incubation of thiol modified antibody onto the gold surface, [55] to provide an electrostatic interaction environment only for the target, two strategies were applied in sequence to maximize the electrostatic interaction.…”

“…[55] Ther ecognition element was target specific antibody.T he transduction element was [Ru(NH 3 ) 6 ] 2+/3+ or [Fe(CN) 6 ] 3À/4À based redox system. Unlike conventional use of redox system as surface impedance indicator, [56] this biosensing strategy applied charged redox system for electrostatically interacting with correspondingly charged protein and further quantification by the electrochemical redox reaction based on the reaction kinetic difference.After incubation of thiol modified antibody onto the gold surface, [55] to provide an electrostatic interaction environment only for the target, two strategies were applied in sequence to maximize the electrostatic interaction. First, based on the charge of the target, the charge of the immunoglobulin Gantibody was neutralized by immobilization of thiol modified poly-arginine or polyglutamic acid onto the gold surface,p roviding an eutral charged environment for target sensing.S econd, the pH condition of the testing environment was altered to trigger the charged condition of the target.…”

“…Thef ormation of biomolecular layer is an essential step for the development of electrochemical biosensing. [45,55,57] Controlled and regulated formation of the biomolecule monolayer is critical and beneficial for electrochemistryguided electron transfer and understanding of surface biomolecular reaction, therefore leading to an enhanced, reproducible high-sensitivity performance for electrochemical biosensor.A ne lectrochemical approach for guided patterning DNAa rray on ag old-sensor surface was developed ( Figure 4A). [58] This controlled formation of the DNA monolayer bio-electrode was then further used to detect human methyltransferase, [59] an enzyme causing DNAm ethylation (further leading to cancer progression).…”

A New Class of Low‐Temperature Plasma‐Activated, Inorganic Salt‐Based Particle‐Free Inks for Inkjet Printing Metals

“…The detection of Aβ 42 requires Aβ 42 antibody immobilization through the formation of 3‐mercaptopropionic acid (MPA) monolayers on the Au film electrode surface. MPA monolayer consists of a thiol group (that can form Au–S bonds) and a carboxylic functional group, which is further processed by N ‐hydroxysuccinimide and 1‐ethyl‐3‐(3‐dimethylaminopropyl carbodiimide to produce an active ester group for the crosslinking of antibodies . DPV was used to measure the faradaic current generated by the redox reaction of [Fe(CN) 6 ] 3‐/4− at different Aβ 42 concentrations (Figure c), which is proportional to the interaction between the redox coupling and different concentrations of surface‐captured antigens as shown in Figure d.…”

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“…A point-of-care system is necessary in resource-limited areas. Thus, simplicity for both the biosensor fabrication and the detection procedures is critical [32]. It is critical to create a reagent-free electrochemical platform which is easy to operate.…”