Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors

We report the investigation of label-free protein detection directly from lysed cells using microcantilever sensors. The integration of an internally referenced microcantilever sensor combined with peptide aptamer technology enables scalable and label-free detection of proteins from a complex biological environment (e.g. cell lysate). The internally referenced microcantilever sensor was found to be effective in minimizing both the effects of thermal drift and non-specific binding interactions with the backside of the cantilever, thereby allowing protein detection in a complex biological background. Highly specific peptide aptamers are used to modify the cantilever surface to specifically detect less than 80nM CDK2 protein from yeast cell lysate. This binding of CDK2 on the microcantilever generates a tensile surface stress of average magnitude equal to 70+/-22mN/m. Similar experiments conducted with quartz crystal microbalance (QCM) technology are consistent with the response observed using microcantilever sensors.     

Microcantilever biosensors

Biosensors are sensors in which biomolecular interactions are used as sensing reactions. Biomolecular interactions, when combined with a microcantilever platform, can produce an extremely powerful biosensing design. The resonance frequency of a microcantilever shifts sensitively due to mass loading from molecular interaction as in the case of any acoustic sensors. In addition, the microcantilevers also undergo bending if the molecular adsorption is confined to a single surface of a microcantilever. This cantilever bending is due to a differential surface stress caused by the forces involved in the adsorption process and is amplified by making the cantilever surfaces chemically different. Lack of specificity, the main disadvantage of the cantilevers, can be overcome by using the extremely selective biochemical reactions such as receptor-ligand, antibody-antigen, or enzyme-substrate reactions. Here we review the microcantilever technology and discuss a number of highly sensitive biochemical sensor applications based on microcantilevers.     

Comparison of a potentiometric and a micromechanical triglyceride biosensor

Sensitive biosensors for detection of triglyceride concentration are important. In this paper we report on two types of silicon based triglyceride sensors: an electrolyte-insulator-semiconductor capacitor (EISCAP) which is a potentiometric device and a polysilicon microcantilever. The detection principle for both sensors is based on the enzymatic hydrolysis of triglyceride though the sensing mechanisms are different: electronic for the EISCAP and mechanical for the microcantilever. The characteristics and performances of the two sensors are critically compared. The EISCAP sensor necessitates the presence of a buffer for stable measurements which limits the sensitivity of the sensor at low concentrations of the bioanalyte to 1mM. The cantilever sensor works without a buffer which improves the lower level of sensitivity to 10 microm. Both sensors are found to give reproducible and reliable results.     

Development of nanomechanical biosensors for detection of the pesticide DDT

We report the use of a novel technique for detection of the organochlorine insecticide compound dichlorodiphenyltrichloroethane (DDT) by measuring the nanometer-scale bending of a microcantilever produced by differential surface stress. A synthetic hapten of the pesticide conjugated with bovine serum albumin (BSA) was covalently immobilised on the gold-coated side of the cantilever by using thiol self assembled monolayers. The immobilisation process is characterised by monitoring the cantilever deflection in real-time. Then specific detection is achieved by exposing the cantilever to a solution of a specific monoclonal antibody to the DDT hapten derivative. The specific binding of the antibodies on the cantilever sensitised side is measured with nanomolar sensitivity. Direct detection is proved by performing competitive assays, in which the cantilever is exposed to a mixed solution of the monoclonal antibody and DDT. The future prospects and limitations to be overcome for the application of nanomechanical sensors for pesticide detection are discussed.     

Investigation of biotin-streptavidin binding interactions using microcantilever sensors

We report the investigation of biotin-streptavidin binding interactions using microcantilever sensors. A symmetric cantilever construction is employed to minimize the effects of thermal drift and the control of surface chemistry on the backside of the cantilever is demonstrated to reduce the effects of non-specific binding interactions on the cantilever. Three structurally different biotin modified cantilever surfaces are used as a model system to study the binding interaction with streptavidin. The cantilever response to the binding of streptavidin on these biotin sensing monolayers is compared. The lowest detection limit of streptavidin using biotin-HPDP is found to be between 1 and 10nM limited by the optical measurement setup. Surface characterization using quartz crystal microbalance (QCM) and high-resolution atomic force microscope (AFM) is used to benchmark the cantilever sensor response. In addition, the QCM and AFM studies reveal that the surface density of bound streptavidin on biotin modified surfaces was low, thereby implying that effects other than steric hindrance are responsible for defining cantilever response.     

Investigation of the antigen antibody reaction between anti-bovine serum albumin (a-BSA) and bovine serum albumin (BSA) using piezoresistive microcantilever based sensors

A new microsensor application based on piezoresistive microcantilever technology has been used to study the interaction of anti-bovine serum albumin (a-BSA) with bovine serum albumin (BSA). A thin layer of BSA attached to a glass slide was used as the active sensing layer for the detection of a-BSA in solution. This design produced a large, consistent cantilever deflection when exposed to the analyte. In this system, the cantilever deflection is measured as a simple resistance change in the piezoresistive channel within the cantilever. In a second set of experiments, 3:1 BSA:PEO protein/polymer blended substrates were used as the active sensing layer for the detection of a-BSA in an aerosol delivery. A distinct signature for the analyte, separate from the water vapor carrier, is obtained for this system.     

Label free novel electrical detection using micromachined PZT monolithic thin film cantilever for the detection of C-reactive protein

In this paper, we report the novel electrical measurement for the label-free detection of C-reactive protein (CRP) using resonant frequency shift in the monolithic thin film cantilever of micromachined Pb(Zr0.52Ti0.48)O3 (PZT) which was fabricated with the composition of SiO2/Ta/Pt/PZT/Pt/SiO2 on silicon nitride (SiNx) supporting layer for the dual purpose of electrical self-excitation and sensing. The specific binding characteristics of CRP antigen to its antibody, which is immobilized with Calixcrown SAMs on Au surface deposited on microcantilever, is determined in high sensitivity to the nanogram level per milliliter by measuring the resonant frequency shift. The nanomechanical PZT cantilever turns out a robust platform for the highly specific antigen-antibody interaction and provides with the novel tool for qualification and quantification of biomolecules without any sample labeling and bulky optical apparatus.     

A potentiometric protein sensor built with surface molecular imprinting method

Surface molecular imprinting, as compared to molecular imprinted bulk polymers, has the advantages of higher re-occupation percentage of the reception sites, fast response, integration of sensing element and transducer, etc. In this study, a potentiometric protein sensor was developed based on the surface molecular imprinting technique. Using the self-assembled monolayers of alkanethiol with hydroxyl terminal groups as the matrix material, and target protein molecules as the template, the sensing layer was created on the surface of the gold-coated silicon chip-an electrochemical transducer. Potentiometric measurement demonstrated that the sensor could selectively detect myoglobin or hemoglobin molecules, either with or without the presence of other protein molecules in the same solution.     

Characterization of QCM sensor surfaces coated with molecularly imprinted nanoparticles

Molecularly imprinted polymers (MIPs) are gaining great interest as tailor-made recognition materials for the development of biomimetic sensors. Various approaches have been adopted to interface MIPs with different transducers, including the use of pre-made imprinted particles and the in situ preparation of thin polymer layers directly on transducer surfaces. In this work we functionalized quartz crystal microbalance (QCM) sensor crystals by coating the sensing surfaces with pre-made molecularly imprinted nanoparticles. The nanoparticles were immobilized on the QCM transducers by physical entrapment in a thin poly(ethylene terephthalate) (PET) layer that was spin-coated on the transducer surface. By controlling the deposition conditions, it was possible to gain a high nanoparticle loading in a stable PET layer, allowing the recognition sites in nanoparticles to be easily accessed by the test analytes. In this work, different sensor surfaces were studied by micro-profilometry and atomic force microscopy and the functionality was evaluated using quartz crystal microbalance with dissipation (QCM-D). The molecular recognition capability of the sensors were also confirmed using radioligand binding analysis by testing their response to the presence of the test compounds, (R)- and (S)-propranolol in aqueous buffer.     

Characterization of immobilization methods for African swine fever virus protein and antibodies with a piezoelectric immunosensor

A direct piezoelectric flow injection analysis immunoassay for the detection of African Swine Fever virus and antibodies is presented. The peptide-specific monoclonal antibody 18BG3 and the virus protein 73 were used for detection with a quartz crystal microbalance. Accumulation of the analyte on the surface of this mass-sensitive biosensor resulted in a shift of the resonant frequency. Highly selective receptor layers were applied on the sensing electrode of the quartz crystal for detection of the complementary analyte. Different immobilization methods proved to be appropriate for coating of the monoclonal antibody 18BG3. A quartz crystal covalently coated with the antibody 18BG3 detected virus protein VP73 samples more than 20 times and was stable for more than 30 days. The coating of virus protein was performed by physisorption. A sensor with a virus protein receptor layer detected antibody 18BG3 samples 10 times within one day. The sensor device was able to perform one measurement cycle including blocking and regeneration within 30 min. With the help of a suitable carrier liquid, measurements with serum samples were performed. The calibration curves for measurements in buffer and in serum could be determined and the detection limits for virus protein detection were 0.31 and 1 μg/ml, and for antibody detection 0.1 and 0.2 μg/ml, respectively.     

A high density microelectrode array biosensor for detection of E. coli O157:H7

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.     

Towards the silicon nanowire-based sensor for intracellular biochemical detection

A microneedle sensor platform with integrated silicon nanowire tip was developed for intracellular biochemical detection. Because of the virtue of miniaturized size and high sensitivity, this sensor has a great potential for studying individual cell or localized bioenvironment by revealing the pH level and/or enzyme activities. The fabrication of the microneedle sensor was primarily based on conventional silicon processing, where a silicon-on-insulator (SOI) wafer with 50 nm thick (100) p-type Si device layer was used as the substrate. The silicon nanowires of 50 nm height and 50-100 nm width were created by electron beam (E-beam) lithography on the tip of microneedle with good electrical connection to the contact pads for convenient electrical measurement. A three layer structure with base, support cantilever, and needle tip was designed to ensure convenient handling of sensors and minimize the invasive penetration into biological cells. In this paper, we demonstrate a preliminary assessment of this novel intracellular sensor with electrical conductance measurement under different pH levels. It is expected that this sensor with proper chemical modification will enable localized biochemical sensing within biological cells, such as neurotransmitter activities during the synaptic communication between neuron cells.     

The use of regenerable, affinity ligand-based surfaces for immunosensor applications.

The regeneration of antibody-binding surfaces is of major importance for re-usable sensor formats such as required for direct 'real-time' biosensing technologies and is often difficult to achieve. Antibodies commonly bind the antigen with high avidity and may themselves be sensitive to regeneration conditions. The interaction of polyclonal anti-chlorpyriphos antibody with an immobilised chlorpyriphos-ovalbumin (chlor-oval) conjugate and the interaction of soluble recombinant CD4 with covalently immobilised anti-CD4 IgG are presented in order to highlight these difficulties. Affinity-capture is suggested as an alternative format as it facilitates surface regeneration, directed immobilisation and the attainment of interaction progress curves that conform to the ideal pseudo-first-order kinetic interaction model. Protein A, protein G and polyclonal anti-mouse Fe-coated surfaces were used to observe the interaction of captured anti-GST monoclonal antibody with glutathione-s-transferase (GST). It was shown that a protein A affinity-capture surface produced ideal interaction progress curves while both protein G and polyclonal anti-mouse Fe resulted in systemic deviations.     

Anti-rabbit immunoglobulin G detection in complex medium by PM-RAIRS and QCM Influence of the antibody immobilisation method

Two antibody immobilisation procedures were compared to set up an immunosensor for goat anti-rabbit immunoglobulin (anti-rIgG), i.e. rIgG covalently bound or immobilised via affinity to protein A (PrA). In both cases, the first layer of protein was covalently bound to a mixed self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) and mercaptohexanol (C6OH) on a gold surface. The elaboration of the sensitive surfaces, as well as their selectivity and sensitivity were studied step by step by polarization modulation-reflection absorption infra-red spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM) with impedance measurement. QCM measurements showed that the viscoelastic properties of the antibody layer were markedly modified during the antigen recognition when the antibody was bound by affinity to PrA. The specific detection of antigen within a complex medium was assessed by PM-RAIRS thanks to the grafting of cobalt-carbonyl probes. Affinity constants between the immobilised rIgG and the anti-rIgG were determined from PM-RAIRS analysis.     

New reactive polymer for protein immobilisation on sensor surfaces

Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ngmL(-1) and for PSA 0.01 ngmL(-1). The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors.     

A Double-Needle Gold-Silver Electrodes Continuous Glucose Monitoring Device

ObjectivesDiabetes is a serious, long-term disease and the use of continuous glucose monitoring sensors can reduce reliance on other painful invasive blood testing methods such as the finger blood glucose test. According to our work, a low-cost continuous glucose sensor has been developed based on electrochemical measurement techniques.MaterialsThe sensor is based on a two needles system; a gold and a silver electrode are integrated into a circular shaped electronic printed circuit board (PCB). The sensing part is based on biological electrochemical measurements. Glucose oxidase (Gox) was used as the active sensing element and ferrocene (Fc) as a mediator. Simple and low-cost coating methods were used; these methods are self-assembled monolayers and deep coating. This will reduce the final cost of the sensor as no expensive technique was used. The electrical subsystem contains a low-noise and low-power trans-impedance front-end as well as a single-chip low-power Bluetooth microcontroller with a 12-bit Analog-to-Digital Converter (ADC).ResultsThe sensor was tested in various concentrations of glucose. As a result of initial in vitro experiments, detailed analytical performance metrics are presented. The device has consistently shown a sensitivity of 3.059 mV/(mg/dl) reading with a linear range of 0-400 mg/dl.ConclusionThe proposed study shows promising results for glucose detection. Thus, this type of sensor can be used for different analyzes targeting biological applications after further investigations and analysis.     

Metal clad leaky waveguides for chemical and biosensing applications

Novel metal clad leaky waveguide (MCLW) sensor devices have been developed for sensing applications. These chips are designed to confine the light in a low refractive index waveguide that encompasses the chemically-selective layer, maximising the overlap between the optical mode and the chemistry, thus improving the sensitivity. In this work, a thin metal layer was inserted between the substrate and the thick waveguide layer, increasing the reflectivity of the waveguide/metal interface and decreasing the light lost at each of reflection in the leaky mode, which in turn increases the propagation distance. The device has been used for a range of biosensing applications, including the detection of organophosphoros pesticides. The limit of detection for paraoxon, based on absorbance detection, was calculated to be 6 nM. Refractive index detection was demonstrated by monitoring the change in the out-coupled angle resulting from the binding of protein A to anti-protein A immobilized on agarose. The sensor was also used for detecting the quenching of the fluorescence of an acid-base sensitive ruthenium complex immobilized within the sol-gel and with glucose oxidase enzyme. The limit of detection for glucose was 3 microM. The advantage of using the metal layer in the MCLW was that an electrical potential could be applied to accelerate the diffusion of the analyte to the immobilised antibody, which resulted in a shortened analysis time and a reduction in non-specific binding.     

Investigation of osteoprotegerin interactions with ligands and antibodies using piezoelectric biosensors

Osteoprotegerin (OPG, osteoclastogenesis inhibitory factor) is a secretory glycoprotein involved as a soluble factor in the regulation of bone mass. OPG and its ligand (RANKL) levels in serum indicate the osteoclast formation activity. Alterations of the RANKL/OPG concentration ratio may be the cause of bone loss in many imbalances including osteoporosis, hypercalcaemia, metastatic osteolytic lesions and rheumatic bone degradation. The interactions of OPG with several antibodies were studied using the piezoelectric quartz crystal sensor. Monoclonal anti-OPG antibodies (5H3, 4E6H9 and OPG1.3) were immobilised on the sensing surface modified with covalently attached monolayer of protein A. Binding of both OPG standard and recombinant OPGFc chimeric protein was followed in real time. All antibodies were able to bind OPG and OPGFc, though in the case of MAb 4E6H9 the immunocomplexes dissociated quickly in the absence of OPG. Alternatively, biorecognition layers with RANKL were used. Two versions of the piezoelectric sensor for OPG were developed. The direct immunosensor was based on the antibody 5H3 and the affinity sensor employed the immobilised RANKL. The RANKL sensor exhibited poor reproducibility of results. For the immunosensor, the measuring range was 1.2-35 U/L of OPG. One analysis was completed within 15 min; the sensors were used repeatedly using regeneration with glycine buffer (pH 2.0). The developed immunosensor seems promising for rapid determination of osteoprotegerin in serum.     

Measurement of mechanical forces generated by plant P-protein aggregates (forisomes)

Mechanical forces generated by forisomes were measured using a microfabricated polymer cantilever sensor. The forces were simultaneously measured in both the longitudinal and radial directions. Sensors were fabricated from polystyrene using the sacrificial layer micromolding process. The sensor response was simulated using finite element analysis. Forces in the longitudinal direction ranged from 84 to 136 nN and forces in the radial direction were 22–61 nN. This device offers a new approach to measuring small magnitude biological forces. In addition, the ability to accurately measure forces generated by forisomes is an important step toward their implementation as functional structures in microdevices.     

Optimisation of glass surfaces for optical immunosensors

The surfaces of glass sensor chips were modified with dextran to generate a layer protecting the sensor surface from unspecific protein binding and also serving as a matrix for covalent protein immobilisation. Dextran was coupled to the glass surface in different concentrations either covalently on amino-functionalised glass chips or via biotin-avidin binding. Unspecific binding of BSA was monitored with the grating coupler system, and was increasingly suppressed with increasing dextran concentrations. Using a solution with 100 mg/ml carboxymethylated dextran decreased the signals to approximately 2% of those obtained at an untreated glass chip. Antibodies were successfully immobilised in the dextran and binding to the corresponding Cy5-labelled antigen was repeatedly monitored using a fluorescence sensor system (total internal reflection fluorescence (TIRF)).