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Integration of biosensing techniques with microfluidics has enabled less sample consumption and increased sensitivity. In addition, it offers a potential improvement with respect to slow diffusion-limited sensing, as well as the incorporation of several sample handling parts into a single lab-on-a-chip device. One highly efficient technique in microfluidics that can be utilized in pre-sensing sample preparation steps such as sorting, separation, trapping, enrichment, and labelling is electrokinetics. AC electroosmosis, AC electrothermal, and dielectrophoresis are among the highly applicable electrokinetic methods used for pre-sensing sample preparation for enhancing sensitivity, limit-of-detection, and selectivity of biosensing devices. In this review article, we focus on these three electrokinetic techniques used for sample enrichment in the field of biosensing technology. First, the working principals of electrothermal, electroosmosis, and dielectrophoresis are characterized, and then their recent advances and major achievements in sample enrichment for biosensors are discussed [ABSTRACT FROM AUTHOR]

The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab’ fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability). [ABSTRACT FROM AUTHOR]

Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab’ fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab’ fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab’ fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab’ fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab’ immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab’ fragments in different applications and future directions of the field will be addressed, especially regarding biosensors. [ABSTRACT FROM AUTHOR]

Single-strand 25-mer oligonucleotides have been attached to the surface of a 9-MHz acoustic wave sensor incorporated into a flow-through configuration. This was achieved by an online interaction of the biotinylated probe with chemisorbed neutravidin. Duplex formation with complementary 25-mer was monitored by acoustic wave detection in real time. Regeneration of the probe-modified surface was achieved using online introduction of λ-exonuclease, an enzyme that digests a single strand of double-stranded DNA beginning with the 5' end, cleaving single nucleotides as it progresses. Thus, with the 5' end of the probe strand remaining inaccessible because of the biotin-neutravidin linkage, the target strand was successfully removed by enzymatic digestion. The results of the acoustic wave work are correlated with analogous experiments involving oligonucleotides labeled with 32P. [ABSTRACT FROM AUTHOR]

The thickness shear mode acoustic wave device is of interest for the sensing of biomarkers for diseases in various biological fluids, but suffers from the issue of non-specific adsorption of compounds other than those of interest to the electrode surface, thus affecting the device's output. The aim of this present study was to determine the level of non-specific adsorption on gold electrodes from serum samples with added ovarian cancer biomarker lysophosphatidic acid in the presence of a surface anti-fouling layer. The latter was an oligoethylene molecule with thiol group for attachment to the electrode surface. It was found that the anti-fouling layer had a minimal effect on the level of both adsorption of components from serum and the marker. This result stands in sharp contrast to the analogous monolayer employed for anti-fouling reduction on silica. [ABSTRACT FROM AUTHOR]

This review will consider the role of biosensors towards the detection of infectious bacteria, although non-infectious ones will be considered where necessary. Recently, there has been a heightened interest in developing rapid and reliable methods of detection. This is especially true for detection of organisms involved in bioterrorism, food poisoning, and clinical problems such as antibiotic resistance. Biosensors can assist in achieving these goals, and sensors using several of the different types of transduction modes are discussed: electrochemical, high frequency (surface acoustic wave), and optical. The paper concludes with a discussion of three areas that may make a great impact in the next few years: integrated (lab-on-a-chip) systems, molecular beacons, and aptamers. [ABSTRACT FROM AUTHOR]

Acoustic wave devices have continued to gain attention as biosensor structures because of their relative ease of operation and sensitivity to interfacial biochemical events. In the present paper, we review the use of the thickness-shear mode device for the label-free detection of processes involving nucleic acid moieties that are imposed at the sensor–liquid interface. Following a concise discussion of the theory and technology connected to the operation of the sensor in liquids, we outline a number of protocols that have been adopted for the attachment of oligonucleotides to sensor surfaces, many of which have been employed in ultrasonic biosensing. The various categories of applications are then surveyed in some detail. By far, the largest group is the study of duplex formation at the sensor surface, involving a compendium of experiments involving complementary and mismatched sequences. Considerably less attention has been paid to the detection of interaction of surface-bound nucleic acids with small molecules such as specific-binding peptides and drugs.A comprehensive appraisal of the literature in this field strongly suggests that acoustic coupling phenomena are particularly sensitive to interfacial physical chemistry. Accordingly, acoustic shear wave technology offers unique advantages over other sensor configurations because of its ability to produce multidimensional information through the recording of various parameters obtained from acoustic network analysis. [Copyright &y& Elsevier]

A critical analysis of optical and acoustic wave instrumentation for examining the transcription apparatus and its regulation is given in the present review. The physico-chemical parameters derived from such in vitro experiments are important from a biophysical standpoint. The overall mechanism of transcription is composed of several mechanisms such as DNA-binding and promoter selection, closed and open polymerase complex formation, initiation of RNA synthesis, elongation and termination. Surface plasmon resonance (SPR) and fluorescence spectroscopy are widely employed techniques for investigating these mechanisms in real time. Although the binding of nucleotides, transcription factors (TFs) and inhibitors to RNA polymerase (RNAP) and the DNA template have been studied extensively, the synthesis of mRNA has not been investigated in detail except by methods based on electrophoresis. The use of acoustic wave physics for investigating transcriptional chemistry offers not only a time-course analysis but also the potential to gain insight into structural changes that occur during the process. [Copyright &y& Elsevier]

[Display omitted] • An affinity-based electrochemical biosensor for early diagnosis of ovarian cancer. • Using stainless steel electrodes to reduce cost and improve sensitivity. • Using silane-based chemistry to overcome non-specific adsorption. • Gelsolin-actin system as the biorecognition surface for detecting LPA. • Proof-of-concept of a label-free biosensor for fabricating a point-of-care device. Electrochemical techniques are considered to be highly sensitive, capable of fast response and can be easily miniaturized, properties which can aid with regard to the fabrication of compact point-of-care medical devices; however, the main challenge in developing such a tool is overcoming a ubiquitous, problematic phenomenon known as non-specific adsorption (NSA). NSA is due to the fouling of non-target molecules in the blood on the recognition surface of the device. To overcome NSA, we have developed an affinity-based electrochemical biosensor using medical-grade stainless steel electrodes and following a unique and novel strategy using silane-based interfacial chemistry to detect lysophosphatidic acid (LPA), a highly promising biomarker, which was found to be elevated in 90 % of stage I OC patients and gradually increases as the disease progresses to later stages. The biorecognition surface was developed using the affinity-based gelsolin-actin system, which was previously investigated by our group to detect LPA using fluorescence spectroscopy. We demonstrate the capability of this label-free biosensor to detect LPA in goat serum with a detection limit of 0.7 µM as a proof-of-concept for the early diagnosis of ovarian cancer. [ABSTRACT FROM AUTHOR]

This paper describes the design and synthesis of 3-(3-(trichlorosilyl)propoxy)propanoyl chloride (MEG-Cl), a compound capable of forming functionalizable monolayers on hydroxylated surfaces. The compound was synthesized in high purity, as suggested by nuclear magnetic resonance analysis, and in moderate overall yield. Contact angle measurement and X-ray photoelectron spectroscopy confirm the binding of MEG-Cl to an amorphous glass substrate and the further modification of the monolayer with a nickel (II)-binding ligand for the purpose of binding polyhistidine-tagged proteins. The compound will be useful in biosensing applications due to its ability to be easily modified with any number of nucleophilic functional groups subsequent to substrate monolayer formation. [ABSTRACT FROM AUTHOR]

Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab’) in biosensors. One Fab’ linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab’ fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab’ fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab’-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection. [ABSTRACT FROM AUTHOR]

Abstract: Herein is described a label-free immunosensor dedicated to the detection of HIV-2. The biosensor platform is constructed as a mixed self-assembled monolayer-coated quartz wafer onto which HIV-2 immunodominant epitopes are immobilized. The biosensing properties, in terms of specific vs. non-specific antigen–antibody interactions, are evaluated with the electromagnetic piezoelectric acoustic sensor (EMPAS) using equimolar serum solutions of HIV-2 or HIV-1 monoclonal antibodies, respectively. This immunosensor constitutes the first real-world application of the EMPAS technology in the bioanalytical field. [Copyright &y& Elsevier]

A concise overview of selected literature concerning biosensors based on surface plasmon resonance and acoustic wave technologies is presented. A comparison in terms of their performance and potential advances in the bioanalytical field is discussed. This Mini-Review highlights the generally underrecognized potential of acoustic wave technology, in the field of biosensors relative to plasmon resonance, and focuses on stimulating not only the development of acoustic wave biosensors but also a broader and increased use of them. These sensors are anticipated to play a central role in biodetection in the near future. [ABSTRACT FROM AUTHOR]

The operation of biosensors requires surfaces that are both highly specific towards the target analyte and that are minimally subject to fouling by species present in a biological fluid. In this work, we further examined the thiosulfonate-based linker in order to construct robust and durable self-assembling monolayers (SAMs) onto hydroxylated surfaces such as silica. These SAMs are capable of the chemoselective immobilization of thiol-containing probes (for analytes) under aqueous conditions in a single, straightforward, reliable, and coupling-free manner. The efficacy of the method was assessed through implementation as a biosensing interface for an ultra-high frequency acoustic wave device dedicated to the detection of avidin via attached biotin. Fouling was assessed via introduction of interfering bovine serum albumin (BSA), IgG antibody, or goat serum. Improvements were investigated systematically through the incorporation of an oligoethylene glycol backbone employed together with a self-assembling diluent without a functional distal group. This work demonstrates that the incorporation of a diluent of relatively short length is crucial for the reduction of fouling. Included in this work is a comparison of the surface attachment of the linker to Si3N4 and AlN, both materials used in sensor technology. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Detection of active plasmin in the 10 nM–32 pM concentration range was achieved. • An electromagnetic piezoelectric acoustic sensor was employed. • Bovine β-casein layers on quartz resonator crystals were used as substrates. • Hydrophobization of quartz crystals led to six times faster β-casein adsorption. • An inverse Michaelis–Menten type equation was used to fit the calibration curve. Abstract Detection of residual plasmin activity in milk represents a difficult challenge for the dairy industry. Conventional methods are either too expensive or incapable of providing enough data from UHT treated milk. Acoustic wave–based biosensors operated in the thickness shear mode (TSM) showed potential for the detection of proteolysis of β-casein, a milk protein by protease plasmin. An ultra-high frequency device, the electromagnetic piezoelectric acoustic sensor (EMPAS), designed to enhance the sensitivity of TSM, was tested for detection of plasmin at low concentrations. β-casein layers immobilised on the hydrophilic or hydrophobized surfaces of EMPAS quartz discs served as substrate for the enzyme. In contrast with conventional TSM devices, the shearing oscillations in EMPAS are induced contactless, by a magnetic coil located 30 μm below the quartz crystal. This configuration allows the registration of unusually high harmonics (up to the 49th-53rd), thus enhancing the sensitivity of detection. On both surface types, the adsorbed β-casein mass and the stability of the layer was compared, with the result that hydrophobic surfaces provide superior conditions for immobilisation than the hydrophilic case. Consequent proteolysis measurements of these substrate layers were carried out in a broad plasmin concentration range (32 pM–10 nM) in flow mode. Initial reaction rates measured at different enzyme concentrations have been used to construct a calibration curve based on an inverse Michaelis–Menten type equation. The sensitivity of the EMPAS allowed measurements of as low as 32 pM concentration of plasmin, reaching (and often exceeding) levels comparable to state of the art techniques like ELISA. The presented method however, unlike ELISA, is effective on a timescale of minutes. [ABSTRACT FROM AUTHOR]

There are currently no biosensors that are able to reliably detect the process of cancer metastasis. We describe the first label-free real-time ultra-high frequency acoustic wave biosensor prototype capable of detecting the breast and prostate cancer metastasis biomarker, parathyroid hormone-related peptide (PTHrP). Two different linkers – 11-trichlorosilyl-undecanoic acid pentafluorophenyl ester (PFP) and S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) – were used to immobilize whole anti-PTHrP antibodies and Fab’ fragments to surfaces as biorecognition elements. The biosensor surfaces were optimized using X-ray photoelectron spectroscopy (XPS) and the ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS). One optimized whole antibody-based surface (PFP/protein G'/whole antibodies/ethanolamine) and one optimized Fab' fragment-based surface (TUBTS/Fab' fragments) were tested as biosensors. It was determined that an in-line injection of bovine serum albumin prior to analyte injection yielded the most minimally fouling surfaces. Each surface was tested with no mass amplification and with sandwich-type secondary antibody mass amplification. The whole antibody-based mass-amplified biosensor yielded the lowest limit of detection (61 ng/mL), highest sensitivity, and a linear range from 61 ng/mL to 100 μg/mL. However, the Fab' fragment-based biosensor displayed better regenerability as a loss of ~20% of the initial analyte signal intensity was observed with each subsequent injection. The whole antibody-based biosensor was only capable of producing an analyte signal in the first injection. [ABSTRACT FROM AUTHOR]

This paper describes a label-free and real-time piezoelectric aptasensor for the detection of cocaine. The acoustic wave sensing platform is a quartz substrate functionalized with an adlayer of S -(11-trichlorosilyl-undecanyl)-benzenethiosulfonate (BTS) cross-linker onto which the anti-cocaine MN4 DNA aptamer is next immobilized. Preparation of the sensor surface was monitored using X-ray photoelectron spectroscopy (XPS), while the binding of cocaine to surface-attached MN4 was evaluated using the electromagnetic piezoelectric acoustic sensor (EMPAS). The MN4 aptamer, unlike other cocaine aptamer variants, has its secondary structure preformed in the unbound state with only tertiary structure changes occurring during target binding. It is postulated that the highly sensitive EMPAS detected the binding of cocaine through target mass loading coupled to aptamer tertiary structure folding. The sensor achieved an apparent K d of 45±12 µM, and a limit of detection of 0.9 µM. Repeated regenerability of the sensor platform was also demonstrated. This work constitutes the first application of EMPAS technology in the field of aptasensors. Furthermore, it is so far one of the very few examples of a bulk acoustic wave aptasensor that is able to directly detect the binding interaction between an aptamer and a small molecule in a facile one-step protocol without the use of a complex assay or signal amplification step. [ABSTRACT FROM AUTHOR]

We report herein three unprecedented alkyltrichlorosilanesurfacemodifiers bearing pentafluorophenyl ester (PFP), benzothiosulfonate(BTS), or novel β-propiolactone (BPL) functionalizable terminalgroups. Evidence is provided that these molecules can be preparedin very high purity (as assessed by NMR) through a last syntheticstep of Pt-catalyzed alkene hydrosilylation then directly employed,without further purification, for the surface modification of quartzand medical grade stainless steel. Subsequent on-surface functionalizationswith amine and thiol model molecules demonstrate the potential ofthese molecular adlayers to be important platforms for future applicationsin the bioanalytical and biomedical fields. [ABSTRACT FROM AUTHOR]

Abstract: The behaviour of proteins on surfaces and at interfaces is an important field with applications in drug development, clinical diagnostics and studies of device biocompatibility. A key factor is the conformation of surface-bound proteins, which can affect chemical signalling and drug binding. A recent study of the interactions of haemoglobin with hydrophobic anions at a liquid–liquid interface has shown that a pH- and orientation-dependent conformational change occurs in the haemoglobin molecule upon interaction with these anions. To corroborate these results, we use an acoustic wave detector to study binding of solution-phase hydrophobic anions to surface-adhered haemoglobin. The orientation of protein is controlled by thiol chemistry, which generates hydrophilic and hydrophobic surfaces. Tetraphenylborate-based anions are introduced to the haemoglobin coated surface via an on-line flow-injection system to monitor the signal in real-time. Changes in the acoustic properties of the surface, measured piezoelectrically, are related to interactions between the protein and the anions. Signal strength is proportional to the degree of interaction between the salts and the haemoglobin, which in turn, is influenced by its conformation. [Copyright &y& Elsevier]

Isolation of neurons from animal tissue is an important aspect of understanding basic biochemical processes such as the action of hormones and neurotransmitters. In the present work, the focus is on an effort to evaluate the utility of acoustic wave physics for the study of such cells. Immortalised hypothalamic neuronal cells from mouse embryos were cultured on the surface of the gold electrode of a 9.0 MHz thickness-shear mode acoustic wave sensor. These cells, which are clonal, are imposed on the surface of the device at a confluence in the range of 80–100%. The coated sensor is incorporated into a flow-injection configuration such that electrolytes can be introduced in order to examine their effects through measurement by network analysis. Both series resonance frequency, fs, and motional resistance, Rm, were measured in a number of experiments involving the injection of KCl and NaCl into the sensor-neuron system. The various responses to these electrolytes were interpreted in terms of changes in cellular structure associated with the depolarization process. The sensor-neuron system was found to elicit different responses to the addition of KCl and NaCl. Preliminary findings indicate that the TSM sensor does not purely measure changes in the membrane potential upon KCl addition. Typical changes in fsfor 15 mM, 30 mM and 60 mM KCl additions were 54 ± 15, 80 ± 26 and 142 ± 58 Hz (mean ± standard deviation) respectively. Typical changes in Rmfor these KCl additions were 7 ± 3, 13 ± 4 and 23 ± 6 Ω, respectively. These results were concluded after 17 runs at each concentration. Despite the large relative standard deviations, the dependence of fsand Rmwith respect to concentration was apparent. Controls performed by coating the TSM sensor with laminin or a cell attachment matrix showed no significant changes in either fsor Rmfor the same solutions tested on the sensor-neuron system. [ABSTRACT FROM AUTHOR]

Abstract: Recent times have seen a rapidly expanding interest in the study of both single cell behaviour and populations of cells. This paper presents a concise review of current techniques employed for the transduction and processing of cellular signals. Among these, electrochemical methodology in the form of transistor and impedance methods has figured prominently. Indirectly connected to this approach has been the optical, light addressable potentiometric technique. In our research we are developing vibrational methods which are capable of examining populations of neurons, smooth muscle and human red blood cells on a substrate in a label-free fashion. These are based on transverse acoustic wave methodology and Kelvin nanoprobe physics. With respect to the former, synchronous oscillations of frequency are detected for neurons which are altered by the introduction of certain drugs. The same technique can be used to monitor chemical perturbation of the structure of smooth muscle cells from rat aorta. The Kelvin nanoprobe possesses sub-micron resolution and has been successfully employed in the characterization of both isolated, single neuron and red blood cells. Alterations in cell behaviour are reflected in apparent changes in work function, which in turn is associated with changes in cellular potential and dielectric properties. [Copyright &y& Elsevier]