Table of Contents

Abstract: The great wealth of different surface sensitive techniques used in biosensing, most of which claim to measure adsorbed mass, can at first glance look unnecessary. However, with each technique relying on a different transducer principle there is something to be gained from a comparison. In this tutorial review, different optical and acoustic evanescent techniques are used to illustrate how an understanding of the transducer principle of each technique can be exploited for further interpretation of hydrated and extended polymer and biological films. Some of the most commonly used surface sensitive biosensor techniques (quartz crystal microbalance, optical waveguide spectroscopy and surface plasmon resonance) are briefly described and five case studies are presented to illustrate how different biosensing techniques can and often should be combined. The case studies deal with representative examples of adsorption of protein films, polymer brushes and lipid membranes, and describe e.g., how to deal with strongly vs. weakly hydrated films, large conformational changes and ordered layers of biomolecules. The presented systems and methods are compared to other representative examples from the increasing literature on the subject.

Abstract: Although still commonly used in clinical practice to screen and diagnose prostate cancer, there are numerous weaknesses of prostate-specific antigen (PSA) testing, including lack of specificity and the inability to distinguish between aggressive and indolent cancers. A promising prostate cancer biomarker, alpha-methylacyl-CoA racemase (AMACR), has been previously demonstrated to distinguish cancer from healthy and benign prostate cells with high sensitivity and specificity. However, no accurate clinically useful assay has been developed. This study reports the development of a single use, disposable biosensor for AMACR detection. Human blood samples were used to verify its validity, reproducibility and reliability. Plasma samples from 9 healthy males, 10 patients with high grade prostatic intraepithelial neoplasia (HGPIN), and 5 prostate cancer patients were measured for AMACR levels. The average AMACR levels in the prostate cancer patients was 10 fold higher (mean(SD) = 0.077 (0.10)) than either the controls (mean(SD) = 0.005 (0.001)) or HGPIN patients (mean(SD) = 0.004 (0.0005)). At a cutoff of between 0.08 and 0.9, we are able to achieve 100% accuracy in separating prostate cancer patients from controls. Our results provide strong evidence demonstrating that this biosensor can perform as a reliable assay for prostate cancer detection and diagnosis.

Abstract: This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format. Polyvinylpyrrolidone (PVP) nanofibers were spun with incorporation of the enzyme horseradish peroxidase (HRP). Scanning electron microscopy (SEM) of the spun nanofibers was used to confirm the non-woven structure which had an average diameter of 155 ± 34 nm. The HRP containing fibers were tested for their change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP by reaction with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. After a storage study over 280 days, 40% of the activity remained. In addition to activity, the fibers were observed to solubilize in the microfluidic chamber. The chromogenic 3,3′,5,5′-tetramethylbenzidine solution reacted immediately with the fibers as they passed through a microfluidic channel. The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform.

Abstract: Angiotensin II is a key regulator of blood pressure and cardiovascular function in mammals. The conversion of angiotensin into its active form is carried out by Angiotensin I-Converting Enzyme (ACE). The measurement of ACE concentration in plasma or serum, its enzymatic activity, and the correlation between an insertion/deletion (I/D) genetic polymorphism of the ACE gene have been investigated as possible indicators of superior athletic performance in humans. In this context, other indicators of superior adaptation to exercise resulting in better athletic performance (such as ventricular hypertrophy, VO₂ max, and competition results) were mostly used to study the association between ACE I/D polymorphism and improved performance. Despite the fact that the existing literature presents little consensus, there is sufficient scientific evidence to warrant further investigation on the usage of ACE activity and the I/D ACE gene polymorphism as biomarkers of superior athletic performance in humans of specific ethnicities or in athletes involved in certain sports. In this sense, a biomarker would be a substance or genetic component that could be measured to provide a degree of certainty, or an indication, of the presence of a certain trait or characteristic that would be beneficial to the athlete’s performance. Difficulties in interpreting and comparing the results of scientific research on the topic arise from dissimilar protocols and variation in study design. This review aims to investigate the current literature on the use of ACE I/D polymorphism as a biomarker of performance in humans through the comparison of scientific publications.

Abstract: Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

Abstract: The detection of pathogenic bacteria remains a challenge for the struggle against biological weapons, nosocomial diseases, and for food safety. In this research, our aim was to develop an easy-to-use electrochemical immunosensor for the detection of pathogenic Staphylococcus aureus ATCC25923. The biosensor was elaborated by the immobilization of anti-S. aureus antibodies using a self-assembled monolayer (SAMs) of 3-Mercaptopropionic acid (MPA). These molecular assemblies were spontaneously formed by the immersion of the substrate in an organic solvent containing the SAMs that can covalently bond to the gold surface. The functionalization of the immunosensor was characterized using two electrochemical techniques: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Here, the analysis was performed in phosphate buffer with ferro/ferricyanide as the redox probe. The EIS technique was used for affinity assays: antibody-cell binding. A linear relationship between the increment in the electron transfer resistance (R_CT) and the logarithmic value of S. aureus concentration was observed between 10 and 10⁶ CFU/mL. The limit of detection (LOD) was observed at 10 CFU/mL, and the reproducibility was calculated to 8%. Finally, a good selectivity versus E. coli and S. epidermidis was obtained for our developed immunosensor demonstrating its specificity towards only S. aureus.

Abstract: Globally, over hundreds of million people are infected with the hepatitis C virus: the global rate of death as a direct result of the hepatitis C virus has increased remarkably. For this reason, the development of efficient drug treatments for the biological effects of the hepatitis C virus is highly necessary. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide can recognize the hepatitis C virus NS3 protein specifically and sensitively. In this study, we elucidated that this biochip can analyze inhibitors to the hepatitis C virus NS3 protein using a nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone demonstrated a remarkable inhibition activity on the hepatitis C virus NS3 protein. Both 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone attenuated the binding affinity in a concentrated manner as evidenced by QDs conjugated RNA oligonucleotide. At a concentration of 0.01 μg·mL⁻¹, 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone showed more than a 30% inhibition activity of a nanoparticle-based RNA oligonucleotide biochip system.

Abstract: This work illustrates the enhancement of the sensitivity of the ELISA titration for recombinant human and native prion proteins, while reducing other non-specific adsorptions that could increase the background signal and lead to a low sensitivity and false positives. It is achieved thanks to the association of plasma chemistry and coating with different amphiphilic molecules bearing either ionic charges and/or long hydrocarbon chains. The treated support by 3-butenylamine hydrochloride improves the signal detection of recombinant protein, while surface modification with the 3,7-dimethylocta-2,6-dien-1-diamine (geranylamine) enhances the sensitivity of the native protein. Beside the surface chemistry effect, these different results are associated with protein conformation.

Abstract: Many cancer treatments rely on inhibition of epidermal growth factor (EGF)-induced cellular responses. Evaluating drug effects on such responses becomes critical to the development of new cancer therapeutics. In this report, we have employed a label-free acoustic sensor, the quartz crystal microbalance with dissipation monitoring (QCM-D), to track the EGF-induced response of mutant MCF10A cells under various inhibitory conditions. We have identified a complex cell de-adhesion process, which can be distinctly altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The dose dependencies of the inhibitors provide IC₅₀ values which are in strong agreement with the values reported in the literature, demonstrating the sensitivity and reliability of the QCM-D as a screening tool. Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions. Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response. Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.

Abstract: An electrotextile with a biosensing focus composed of conductive polymer coated microfibers that contain functional attachment sites for biorecognition elements was developed. Experiments were conducted to select a compound with a pendant functional group for inclusion in the polymer, a fiber platform, and polymerization solvent. The effects of dopant inclusion and post-polymerization wash steps were also analyzed. Finally, the successful attachment of avidin, which was then used to capture biotin, to the electrotextile was achieved. The initial results show a nonwoven fiber matrix can be successfully coated in a conductive, functionalized polymer while still maintaining surface area and fiber durability. A polypropylene fiber platform with a conductive polypyrrole coating using iron (III) chloride as an oxidant, water as a solvent, and 5-sulfosalicylic acid as a dopant exhibited the best coating consistency, material durability, and lowest resistance. Biological attachment of avidin was achieved on the fibers through the inclusion of a carboxyl functional group via 3-thiopheneacetic acid in the monomer. The immobilized avidin was then successfully used to capture biotin. This was confirmed through the use of fluorescent quantum dots and confocal microscopy. A preliminary electrochemical experiment using avidin for biotin detection was conducted. This technology will be extremely useful in the formation of electrotextiles for use in biosensor systems.