Implementation and Characterization of a Fully Miniaturized Biosensor for Endotoxin Detection Based on Electrochemical Techniques

Graphene-based electrochemical biosensors have emerged as promising tools for the early detection and monitoring of oral cancer through salivary biomarker analysis. Graphene's exceptional properties, including high surface area, superior electrical conductivity, and excellent mechanical strength, enable the development of highly sensitive and specific biosensors. This review provides a comprehensive overview of the current state-of-the-art in graphene-based electrochemical biosensors for salivary biomarker detection in oral cancer. We discuss the unique advantages of saliva as a diagnostic medium and highlight the key salivary biomarkers associated with oral cancer, including proteins, DNA, and RNA. Various electrochemical detection techniques, such as cyclic voltammetry, differential pulse voltammetry, electrochemical impedance spectroscopy, field-effect transistors, amperometry, chronoamperometry, and photoelectrochemical methods, are explored in the context of graphene-based biosensors. The challenges associated with the development and clinical translation of these biosensors are also addressed, emphasizing the need for improved functionalization strategies, enhanced stability, and standardized validation protocols. Finally, we present a futuristic outlook on the integration of graphene-based biosensors with artificial intelligence, microfluidics, and telemedicine platforms to enable personalized diagnostics and treatment monitoring. With continued advancements in sensor technology and computational tools, graphene-based electrochemical biosensors have the potential to revolutionize oral cancer management, improving patient outcomes and quality of life. • Offer sensitive detection of salivary biomarkers. • Saliva is a non-invasive diagnostic medium. • Employ various electrochemical techniques. • Challenges include complex cancer biology, low biomarker concentrations, and individual saliva composition variability. • Future advancements include integration with AI and microfluidics.

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

An electrochemical DNA biosensor for the detection of carcinoembryonic antigen-related cell adhesion molecule (CEACAM5), a specific tumor marker for colorectal cancer, is presented. The biosensor based on microelectronicmechanical systems (MEMS) technology, consists of a three electrode configuration where the working electrode is a gold thin-film. The immobilization of the previously designed DNA capture probe is performed by self-assembled monolayer (SAM) technique, and it is hybridized with the complementary target. The formation of the biorecognition surface (SAM) and the detection of the hybridization event are characterized by two different electrochemical techniques: Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV), using potassium ferricyanide/ferrocyanide as redox couple. The differences in the charge transfer resistance (Rct), capacitance (C), and in the redox signals of the potassium ferricyanide/ferrocyanide upon the hybridization, show that the biosensor presented is a rapid, low-cost, and effective tool for CEACAM5 detection. Moreover, the specificity in the hybridization event is demonstrated by exposing the biorecognition surface to a noncomplementary target.

Cyclic voltammetry and electrochemical impedance spectroscopy were used to study the surface acid‐base property of carboxylic acid‐terminated self‐assembled monolayers (SAMs). A carboxylic acid‐terminated thiol, such as thioctic acid (l,2‐dithiolane‐3‐pentanoic acid), was self‐assembled on gold electrodes. Electron transfer between the bulk solution and the SAM modified electrode was studied at different pH using Fe(CN)63− as a probe. The surface pKa. of thioctic acid was determined by cyclic voltammetry and electrochemical impedance spectroscopy to be 5.6 ± 0.1 and 5.8 ± 0.1, respectively. The method is compared with other methods of monolayer pKa measurement.

Probing cysteine self-assembled monolayers over gold nanoparticles – Towards selective electrochemical sensors

Self-assembled monolayer(SAM)로 변형된 금 전극 위로 폴리피롤의 전기화학적 석출 과정을 수용액 상태에서 in-situ EQCM (Electrochemical Quartz Crystal Microbalance)과 ex-situ AFM (Atomic Force Microscopy)을 이용하여 조사하였다. 금 전극에서 cyclic voltammetry로 살펴본 폴리머의 석출은 산화 제한 전위 (anodic limiting potential) 값에 매우 의존적이었으며 주사 횟수에는 의존하지 않았다. 제한 산화 전위가 0.8V (vs Ag | ArCl) 이상일 때 폴리머의 석출은 크게 증가하였다. 그리고 주사 횟수가 증가하면서 질량의 비이상적 변화가 관찰되었는데 이것은 폴리피롤 필름의 rearrangement가 원인이라고 생각된다. 1-dodecanethiol SAM 전극과 thiophene SAM전극에서는 폴리머가 3차원적으로 성장하며 필름의 rearrangement를 수반하였지만 BPUS <TEX>$(Bis(\omega(N-pyrrolyl)-n-undecyl)disulfide)$</TEX> SAM 전극에서는 2차원적인 layer-by-layer 성장을 하고 필름의 rearrangement는 관찰되지 않았다. 폴리머가 급격하게 전극 면으로 석출되면 사슬 모양과 도너츠 모양의 폴리머를 만들며, 정류 상태에 이르면서 주름잡힌 폴리머 필름이 생성되는 것이 원자 힘 현미경 (Atomic Force Microscopy) 이미지로 관찰되었다. The growth of Polypyrrole film has been investigated during electropolymerization in an aqueous solution on bare and SAM modified gold electrodes by in-situ EQCM and ex-situ AFM. According to the result of cyclic voltammetry measurements, in the case of a bare gold electrode, the electrochemical deposition of polypyrrole were dependent on the limiting oxidative potential, but not on scan numbers. When the limiting potential higher than 0.8 V was applied on the electrode, the amount of polypyrrole deposited on a gold electrode was rapidly increased and the abnormal mass change attributed to the rearrangement of polypyrrole films was observed as the scan number increased. The polypyrrole film Prepared on electrodes modified with 1-dodecanethiol SAM or thiophene SAM grew 3-dimensionally with the rearrangement of film. However, in the case of BPUS SAM, 2-dimensional layer-by-layer growth of film was observed without the rearrangement of film. AFM images showed films with chain-shaped and/ or donut-shaped polymers when grown rapidly and a wrinkled film at the steady state condition.

In the present study, we have analyzed the real human plasma effect for the detection of the cytokine biomarker; interleukin-10 (IL- 10) using an electrochemical impedance spectroscopy (EIS) based biosensor. IL-10 is one of the many antigens that are secreted in acute stages of inflammation after left ventricle assisted device (LVAD) implantation for patients suffering from heart failure (HF). For this interest, a biosensor was developed in order to increase the sensitivity of detection in complex medium and in the presence= of other interferences: interleukin-6 (IL-6) and interleukin-1 (IL-1) that are also secreted after LVAD implantation. The anti-human IL-10 monoclonal antibodies (anti-IL-10 mAb’s) were immobilized onto gold microelectrodes through functionalization with selfassembled monolayers (SAMs) of 16-Mercaptohexadecanoic acid b(MHDA) followed by carbodiimide chemistry. Cyclic voltammetry (CV) was applied during the microelectrode functionalization process to characterize the gold microelectrode surface properties. EIS analysis was made for studying the human plasma effect and for cytokine detection. The biosensor was highly selective and sensitive toward the corresponding cytokines IL-10. These were detected in real human plasma samples diluted 1000-fold, by using EIS: before LVAD implantation, after 24 hrs, and after 72 hrs of LVADs implantation at 16.9 ± 1.5 pg/mL, 62.4 ± 2.6 pg/ mL, and 37.6 ± 6.2 pg/mL, respectively. IL-10 was also detected by enzyme-linked immunosorbent assay (ELISA) using the same plasma samples for the same periods of LVAD implantation at 11.3 pg/mL, 57.2 pg/mL, and 34.0 pg/mL, respectively. The present electrochemical biosensor is very promising for an alternative, rapid, low cost, and sensitive technique for cytokine detection that could be at the patient’s bedside using point-of-care testing (POCT). This can provide rapid quantitative information to clinicians and doctors in order to predict the first signs of inflammation after LVAD implantation.

Self-assembled monolayers (SAMs) are widely used as surface modification structures for thin films synthesis and designed nanostructures fabrication1. Alkyltrichlorosilanes is a group of SAMs widely applied in the field of area-selective atomic layer deposition (AS-ALD). The closely packed octadecyltrichlorosilane (ODTS) SAMs could act as monolayer resist to prohibit ALD2, while the pinholes on unsaturated ODTS SAMs could act as nucleation sites to initiate ALD growth. Thus the distribution and structure of pinholes, including its size and density, are important factors for utilizing SAMs template to fabricate nanoparticles via ALD. Many studies characterize SAMs and pinholes by atomic force microscopy (AFM), scanning electron microscopy (SEM), scanning tunneling microscope (STM), etc3. Most of these methods could provide the microscopic information on the pinhole structures, yet the information from these methods is quite localized and hard to obtain statistical average and full-scale information about the pinhole size and distribution density. Here we report utilizing electrochemical impedance spectroscopy (EIS) to study the size distribution and loading of initial nanoparticles grown in those pinhole sites. EIS is a powerful and surface sensitive characterization technique to obtain the average and global characters of the detected surfaces4,5. In this study, the properties of unsaturated ODTS SAMs are characterized with EIS methods. The relationship between SAMs growth time and the pinholes properties has been established. With the SAMs immersing time increasing, the sizes and density distribution are both reduced. For SAMs sample immersed over long time, at least 24 hours, negligible pinholes are observed. Furthermore, Pd ALD process has been carried on those SAMs modified substrates. The distribution of Pd nanoparticles corresponds well with the pinholes. The results show that the nanoparticles grown on SAMs modified substrates depend on the pinholes properties. With SAMs modified substrate, ALD grown noble metal nanoparticles with uniform size and controlled loading density can be achieved.

A self‐assembled monolayer (SAM) modified by 4‐formylphenylboronic acid was formed on the gold electrode, which was applied for the determination of trace concentrations of Cu(II). The formation of advanced SAM on the gold electrode was evidenced by electrochemical impedance spectroscopy, atomic force microscopy and contact angle measurements. Electrochemical determination of Cu(II) ions was performed by square wave voltammetry. Some mutual interferences caused by Cd(II), Co(II), Fe(II), Ni(II) and Pb(II) ions were investigated and it was demonstrated how the negative effects of these interfering ions could be eliminated by adjustment of proper parameters of square wave voltammetry.

Effect of glassy carbon, gold, and nickel electrodes on nickel electrocrystallization in an industrial electrolyte

Norovirus (NoV) infection is one of the most common non-bacterial causes of gastroenteritis among the population worldwide. From the point of view of medical diagnostics, it is important to develop a system that would sensitively and selectively detect norovirus from a patient's sample in order to control and limit its spread. In this paper, we present a stable and sensitive NoV (mouse model) detection matrix in infected food samples. The bio-platform was made of a modified gold electrode with a self-assembled l-cysteine monolayer, covered with gold nanoparticles, a linker and an antibody specific to the VP1 surface protein of the virus. Binding of the VP1 protein to the antibody caused a decrease in the current strength confirmed by electrochemical techniques - cyclic voltammetry (CV) and differential pulse voltammetry. The reduction of the current was proportional to the concentration of NoV sample. The biosensors showed high sensitivity and linearity in a range from 1 × 10-9 to 1 × 10-18 TCID50, with the detection limit of 1 × 10-18 TCID50. CV showed a diffusion-controlled process. In addition, each modification step was confirmed by scanning electron microscopy, electrochemical impedance spectroscopy, and CV. The described immunosensor showed excellent recovery values, good linearity and long-term stability, crucial parameters for biosensor construction.

An amperometric pesticide biosensor has been devised by the composite assembly of silver nanoparticles with avidin and biotinylated acetylcholinesterase (AChE) on gold electrodes modified with a biotin‐terminated self assembly monolayer (SAM). This composite assembly strategy takes use of the biospecific recognition avidin with the biotin from the SAM‐terminals and biotinylated AChE, as well as the electrostatic interaction between silver nanoparticles with negatively charged citrate shell and avidin with encounter charge at pH 7.2. The construction process of the composite interface on gold was monitored by surface plasmon resonance (SPR), and its structure was characterized by attenuated total reflection Fourier‐transform infrared spectra, atomic force microscopy and UV‐vis spectra. The composite interface shows excellent electron transfer ability, as characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Under the optimum conditions a quantitative measurement of organophosphate pesticide dimethoate was achieved with the linear range of 0.05 μM to10 μM and the detection limit 0.01 μM, taken as the concentration equivalent to a 10% decrease in signal. Silver nanoparticles conjugated biotin‐avidin system represents a simple and functional approach to the integration of electrode sensing interface with improved biocompatibility and electron transfer ability, which may provide an analytical access to a large group of enzymes for bioelectrochemical application.

Alkanethiol Self-Assembled Monolayers (SAMs), typically deposited on Au substrates, have been commonly utilized to immobilize receptor biomolecules in the construction of a variety of biosensors, such as the detection of glucose oxidase by the immobilization of calixarenes thiols 1. The appeal of using SAMs derives from their ease of fabrication and component flexibility, allowing for more than one alkanethiol to be deposited in a single monolayer. This results in the formation of SAMs in which each thiol component adds a different property or role, thus introducing versatility to the construction and operation of resulting sensors 2.In our recent work, alkanethiol SAMs, composed of three different thiol molecules, were constructed for use as an electrochemical Toll-Like Receptor 4 (TLR4)-based sensor that can detect Gram-negative bacteria. TLRs are a family of immunoproteins in the innate immune system, which are able to detect a broad category of pathogens, rather than specific targets, as is the case with the adaptive system (e.g., antibodies). In our published work 3, SAMs consisted of a carboxylic acid terminated thiol that allowed for the conversion to a nitrilotriacetic (NTA) group. This moiety could then chelate a Ni2+ cation, followed by attachment of the TLR4 immunoprotein via its His-tag. The second thiol in the SAM contained a terminal ferrocene (Fc) group, where the Fc group was bound to the C11 chain via an amide group. Here, the Fc group serves as a mediator for charge transfer between ferrocyanide in solution and the Au substrate, resulting in relatively high current signals, thus making the sensor potentially field deployable. The third thiol in these SAMs was a hydroxy-terminated thiol, added as a diluent between the immobilized TLR4 receptors. SAMs were prepared by co-deposition on sputter Au on glass.Electrochemical testing involved cyclic voltammetry (CV) studies in pH 7 buffer solution in order to determine the amount of Fc-thiol present, as well as CV and electrochemical impedance spectroscopy (EIS) studies in ferrocyanide solutions to examine its redox-mediation response and to obtain a measure of the charge transfer rate. In the presence of lysed bacteria, the immobilized TLR4 dimerizes, which blocks electrochemical mediation between ferrocyanide and the surface bound Fc. This results in an increase in RCT which is our biosensor signal. However, a key problem with these sensors is that their RCT increased with electrochemical testing in the absence of lysed bacteria, producing signal drift. Thus, in the present work, the reasons for sensor signal drift and methods to overcome it were explored.It was found in recent work that the drift of the full TLR4 sensor was correlated with the loss in the Fc redox signal, although it was not clear if underlying SAM was at the root of the problem. If so, then it was necessary to determine if the loss in the Fc redox activity was caused by the physical loss of the Fc thiol from the Au surface or deactivation of the Fc surface complex and/or its electrochemistry. To determine which of these options was responsible for signal drift, the relative % v/v fraction of the three thiols was varied, and in later work, based on chemical considerations, the Fc molecule containing an amide group was replaced with a C11 chain bonded directly to the Fc. It was found that this latter strategy led to a very stable SAM, showing negligible drift in the Fc CV charges and in the RCT values in ferrocyanide solutions, with or without the TLR4 receptor attached on the outer surface of the SAM.This presentation will focus on presenting the electrochemistry of the various binary and ternary thiol SAMs investigated in this work and examining the Fc CV peak charges, potential, and kinetics, as well as the double layer capacitance, to better understand how and why the Fc redox response was stabilized. This is important because this correlates directly with the stability of the TLR4/Gram-negative bacteria sensor, as will be demonstrated for a TLR2 biosensor tested against Gram-positive bacteria cell wall ligands.

Two anion receptors, 1 and 2, based on the calix[6]crown-4 architecture were synthesized and characterized by NMR (1H, 13C, COSY), UV-vis, and MALDI-MS. 1H NMR measurements demonstrate that receptors 1 and 2 exhibit the highest binding affinity for fluoride ions compared to other anions including Cl-, Br-, NO3-, HSO4-, H2PO4-, and AcO-. The binding constants of 1 with F- and AcO- are 326 (+/-32) and 238 (+/-23) M-1, whereas those of 2 with F- and AcO- are 222 (+/-25) and 176 (+/-21) M-1. The fluorescent titration of 2 with various anions such as Cl-, Br-, NO3-, HSO4-, and H2PO4- led to essentially no change in excimer emission and a slight enhancement of monomer emission. In contrast, a dramatic change was observed in the fluorescence spectra upon the addition of F- and AcO- to 2. Self-assembled monolayers (SAMs) of 1 were formed on gold surfaces and characterized by reductive desorption and other techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy were used to monitor anion recognition by the SAM-modified gold electrodes. The gold electrodes modified by SAMs of 1, upon binding with the F- anion, exhibit a dramatic increase in charge-transfer resistance (Rct) values. This is due to the repulsion between the negatively charged electrode surfaces and the negatively charged Fe(CN)6(3-/4-) redox probe in the electrolyte solution. In contrast, smaller increases in Rct values were observed in the cases of other monovalent anions investigated.

Electron transfer studies on cholesterol LB films assembled on thiophenol and 2-naphthalenethiol self-assembled monolayers