Development of Electrochemical Immunosensors for HER-1 and HER-2 Analysis in Serum for Breast Cancer Patients.

In this work, two human epidermal growth factor receptors, HER-1 and HER-2, were selected as biomarkers to enable the detection of breast cancer. Therefore, two biosensors were developed using gold sensor chips coupled with amperometric detection of the enzyme label horse radish peroxidase (HRP). The biosensors/immunosensors relied on indirect sandwich enzyme-linked immunosorbent assays with monoclonal antibodies (Ab) against HER-1 and HER-2 attached to the sensors to capture the biomarkers. Detection polyclonal antibodies followed by secondary anti-rabbit (for HER-1) and anti-goat (for HER-2) IgG antibody-HRP were then applied for signal generation. In buffer, the developed sensors showed limits of detections (LOD) of 1.06 ng mL-1 and 0.95 ng mL-1 and limits of quantification (LOQ) of 2.1 ng mL-1 and 1.5 ng mL-1 for HER-1 and HER-2, respectively. In 100% (undiluted) serum, LODs of 1.2 ng mL-1 and 1.47 ng mL-1 and LOQs of 1.5 ng mL-1 and 2.1 ng mL-1 were obtained for HER-1 and HER-2, respectively. Such limits of detections are within the serum clinical range for the two biomarkers. Furthermore, gold nanoparticles (AuNP) labelled with secondary anti-rabbit and anti-goat IgG antibody-HRP were then used to enhance the assay signal and increase the sensitivity. In buffers, LODs of 30 pg mL-1 were seen for both sensors and LOQs of 98 pg mL-1 and 35 pg mL-1 were recorded for HER-1 and HER-2, respectively. For HER-2 the AuNPs biosensor was also tested in 100% serum obtaining a LOD of 50 pg mL-1 and a LOQ of 80 pg mL-1. The HER-2 AuNP electrochemical immunosensor showed high specificity with very low cross-reactivity to HER-1. These findings demonstrate that the two developed sensors can enable early detection as well as monitoring of disease progression with a beneficial impact on patient survival and clinical outcomes.

A Comparison of EIS and QCM NanoMIP-Based Sensors for Morphine.

In this work we have compared two different sensing platforms for the detection of morphine as an example of a low molecular weight target analyte. For this, molecularly imprinted polymer nanoparticles (NanoMIP), synthesized with an affinity towards morphine, were attached to an electrochemical impedance spectroscopy (EIS) and a quartz crystal microbalance (QCM) sensor. Assay design, sensors fabrication, analyte sensitivity and specificity were performed using similar methods. The results showed that the EIS sensor achieved a limit of detection (LOD) of 0.11 ng·mL-1, which is three orders of magnitude lower than the 0.19 µg·mL-1 achieved using the QCM sensor. Both the EIS and the QCM sensors were found to be able to specifically detect morphine in a direct assay format. However, the QCM method required conjugation of gold nanoparticles (AuNPs) to the small analyte (morphine) to amplify the signal and achieve a LOD in the µg·mL-1 range. Conversely, the EIS sensor method was labor-intensive and required extensive data handling and processing, resulting in longer analysis times (~30-40 min). In addition, whereas the QCM enables visualization of the binding events between the target molecule and the sensor in real-time, the EIS method does not allow such a feature and measurements are taken post-binding. The work also highlighted the advantages of using QCM as an automated, rapid and multiplex sensor compared to the much simpler EIS platform used in this work, though, the QCM method will require sample preparation, especially when a sensitive (ng·mL-1) detection of a small analyte is needed.

Molecularly Imprinted Nanoparticles Based Sensor for Cocaine Detection.

The development of a sensor based on molecularly imprinted polymer nanoparticles (nanoMIPs) and electrochemical impedance spectroscopy (EIS) for the detection of trace levels of cocaine is described in this paper. NanoMIPs for cocaine detection, synthesized using a solid phase, were applied as the sensing element. The nanoMIPs were first characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering and found to be ~148.35 ± 24.69 nm in size, using TEM. The nanoMIPs were then covalently attached to gold screen-printed electrodes and a cocaine direct binding assay was developed and optimized, using EIS as the sensing principle. EIS was recorded at a potential of 0.12 V over the frequency range from 0.1 Hz to 50 kHz, with a modulation voltage of 10 mV. The nanoMIPs sensor was able to detect cocaine in a linear range between 100 pg mL-1 and 50 ng mL-1 (R2 = 0.984; p-value = 0.00001) and with a limit of detection of 0.24 ng mL-1 (0.70 nM). The sensor showed no cross-reactivity toward morphine and a negligible response toward levamisole after optimizing the sensor surface blocking and assay conditions. The developed sensor has the potential to offer a highly sensitive, portable and cost-effective method for cocaine detection.

Biosensing the Histamine Producing Potential of Bacteria in Tuna.

Histamine poisoning is the most common cause of human foodborne illness due to the consumption of fish products. An enzyme-based amperometric biosensor was developed to be used as a screening tool to detect histamine and histamine-producing bacteria (HPB) in tuna. It was developed by immobilizing histidine decarboxylase and horseradish peroxidase on the surface of screen-printed electrodes through a cross-linking procedure employing glutaraldehyde and bovine serum albumin. The signal generated in presence of histamine at the surface of the electrode was measured by chronoamperometry at in presence of a soluble redox mediator. The sensitivity of the electrode was 1.31-1.59 µA/mM, with a linear range from 2 to 20 µg/ml and detection limit of 0.11 µg/ml. In this study fresh tuna filets purchased in supermarkets in different days (n = 8) were analyzed to detect HPB. Samples with different concentration of histamine were analyzed with culture-based counting methods, biosensor and HPLC and also a challenge test was made. Recovery of histamine from cultures and tuna samples was also assessed. The presence of Morganella psychrotolerans, Photobacterium phosphoreum, P. damselae and Hafnia alvei was detected using culture- and PCR-based methods. At the time of purchase these tuna samples had histamine concentrations from below the limit of detection (LOD) to 60 µg/g. HPLC and biosensor methods provided similar results in the range from zero to 432 µg/g (correlation coefficient, R 2 = 0.990) and the recovery of histamine from cultures and tuna samples was very high (mean bias -12.69 to 1.63%, with root-mean-square error <12%). These results clearly show that fresh tuna is commonly contaminated with strong HPB. The histamine biosensor can be used by the Food Business Operators as a screening tool to detect their presence and to determine whether their process controls are adequate or not.

Subtractive inhibition assay for the detection of Campylobacter jejuni in chicken samples using surface plasmon resonance.

In this work, a subtractive inhibition assay (SIA) based on surface plasmon resonance (SPR) for the rapid detection of Campylobacter jejuni was developed. For this, rabbit polyclonal antibody with specificity to C. jejuni was first mixed with C. jejuni cells and unbound antibody was subsequently separated using a sequential process of centrifugation and then detected using an immobilized goat anti-rabbit IgG polyclonal antibody on the SPR sensor chip. This SIA-SPR method showed excellent sensitivity for C. jejuni with a limit of detection (LOD) of 131 ± 4 CFU mL-1 and a 95% confidence interval from 122 to 140 CFU mL-1. The method has also high specificity. The developed method showed low cross-reactivity to bacterial pathogens such as Salmonella enterica serovar Typhimurium (7.8%), Listeria monocytogenes (3.88%) and Escherichia coli (1.56%). The SIA-SPR method together with the culturing (plating) method was able to detect C. jejuni in the real chicken sample at less than 500 CFU mL-1, the minimum infectious dose for C. jejuni while a commercial ELISA kit was unable to detect the bacterium. Since the currently available detection tools rely on culturing methods, which take more than 48 hours to detect the bacterium, the developed method in this work has the potential to be a rapid and sensitive detection method for C. jejuni.

An immunosensor for parasite lactate dehydrogenase detection as a malaria biomarker - Comparison with commercial test kit.

This paper describes the development of an affinity sensor for the detection of Plasmodium falciparum parasite lactate dehydrogenase (pLDH) as one of the biomarkers used for malaria detection. The gold sensor was functionalised with anti-pLDH after cleaning the electrode surface to remove impurities (120 °C, 1 h). The sensor was then treated to block unreacted groups on the surface and minimise matrix interference, before applying it in a sandwich assay to detect pLDH in buffer samples using a dose concentration assay. The sensor was optimised to achieve the best detection sensitivity before using it for pLDH detection in serum samples. The developed sensor achieved a limit of detection (LOD) of 1.80 ng mL-1 and 0.70 ng mL-1 for the detection of pLDH in buffer and in serum samples respectively. The sensor sensitivity was enhanced further with the use of AuNP conjugated to the detection anti-pLDH-enzyme, achieving an LOD of 19 pg mL-1 in buffer and 23 pg mL-1 in serum samples. The performance of the sensor was compared to commercially available Plasmodium immunochromatographic (ICT) malaria kits. The developed sensor was able to detect pLDH in the Dd2luc culture medium supernatant at 0.002% parasitaemia without the use of AuNP signal enhancement when compared to the OptiMAL-IT ICT kit (detect pLDH) and the BinaxNOW ICT kit (detection of both pLDH and PfHRP 2) samples. Therefore, the sensor developed in this work is highly sensitive and can be used for pLDH detection for on-site diagnosis of malaria. A cheap and simple device as developed in this work is required to tackle malaria detection.

Development of a ß-Lactoglobulin Sensor Based on SPR for Milk Allergens Detection.

A sensitive and label-free surface plasmon resonance (SPR) based sensor was developed in this work for the detection of milk allergens. ß-lactoglobulin (BLG) protein was used as the biomarker for cow milk detection. This is to be used directly in final rinse samples of cleaning in-place (CIP) systems of food manufacturers. The affinity assay was optimised and characterised before a standard curve was performed in pure buffer conditions, giving a detection limit of 0.164 µg mL-1 as a direct binding assay. The detection limit can be further enhanced through the use of a sandwich assay and amplification with nanomaterials. However, this was not required here, as the detection limit achieved exceeded the required allergen detection levels of 2 µg mL-1 for ß-lactoglobulin. The binding affinities of the polyclonal antibody for BLG, expressed by the dissociation constant (KD), were equal to 2.59 × 10-9 M. The developed SPR-based sensor offers several advantages in terms of label-free detection, real-time measurements, potential on-line system and superior sensitivity when compared to ELISA-based techniques. The method is novel for this application and could be applied to wider food allergen risk management decision(s) in food manufacturing.

Synthesis of Molecularly Imprinted Polymer Nanoparticles for α-Casein Detection Using Surface Plasmon Resonance as a Milk Allergen Sensor.

Food recalls due to undeclared allergens or contamination are costly to the food manufacturing industry worldwide. As the industry strives for better manufacturing efficiencies over a diverse range of food products, there is a need for the development of new analytical techniques to improve monitoring of the presence of unintended food allergens during the food manufacturing process. In particular, the monitoring of wash samples from cleaning in place systems (CIP), used in the cleaning of food processing equipment, would allow for the effective removal of allergen containing ingredients in between food batches. Casein proteins constitute the biggest group of proteins in milk and hence are the most common milk protein allergen in food ingredients. As such, these proteins could present an ideal analyte for cleaning validation. In this work, molecularly imprinted polymer nanoparticles (nanoMIPs) with high affinity toward bovine α-casein were synthesized using a solid-phase imprinting method. The nanoMIPs were then characterized and incorporated into label free surface plasmon resonance (SPR) based sensor. The nanoMIPs demonstrated good binding affinity and selectivity toward α-casein (KD ∼ 10 × 10-9 M). This simple affinity sensor demonstrated the quantitative detection of α-casein achieving a detection limit of 127 ± 97.6 ng mL-1 (0.127 ppm) which is far superior to existing commercially available ELISA kits. Recoveries from spiked CIP wastewater samples were within the acceptable range (87-120%). The reported sensor could allow food manufacturers to adequately monitor and manage food allergen risk in food processing environments while ensuring that the food produced is safe for the consumer.

Development of an Immunosensor for PfHRP 2 as a Biomarker for Malaria Detection.

Plasmodium falciparum histidine-rich protein 2 (PfHRP 2) was selected in this work as the biomarker for the detection and diagnosis of malaria. An enzyme-linked immunosorbent assay (ELISA) was first developed to evaluate the immunoreagent's suitability for the sensor's development. A gold-based sensor with an integrated counter and an Ag/AgCl reference electrode was first selected and characterised and then used to develop the immunosensor for PfHRP 2, which enables a low cost, easy to use, and sensitive biosensor for malaria diagnosis. The sensor was applied to immobilise the anti-PfHRP 2 monoclonal antibody as the capture receptor. A sandwich ELISA assay format was constructed using horseradish peroxidase (HRP) as the enzyme label, and the electrochemical signal was generated using a 3, 3', 5, 5'tetramethyl-benzidine dihydrochloride (TMB)/H2O2 system. The performance of the assay and the sensor were optimised and characterised, achieving a PfHRP 2 limit of detection (LOD) of 2.14 ng·mL-1 in buffer samples and 2.95 ng∙mL-1 in 100% spiked serum samples. The assay signal was then amplified using gold nanoparticles conjugated detection antibody-enzyme and a detection limit of 36 pg∙mL-1 was achieved in buffer samples and 40 pg∙mL-1 in serum samples. This sensor format is ideal for malaria detection and on-site analysis as a point-of-care device (POC) in resource-limited settings where the implementation of malaria diagnostics is essential in control and elimination efforts.

The use of differential scanning fluorimetry in the rational design of plastic antibodies for protein targets.

The development of molecularly imprinted polymer nanoparticles (MIP-NPs), which specifically bind biomolecules, is of great interest in the area of biosensors, sample purification, therapeutic agents and biotechnology. Polymerisation techniques such as precipitation polymerisation, solid phase synthesis and core shell surface imprinting have allowed for significant improvements to be made in developing MIP-NPs which specifically recognise proteins. However, the development of MIP-NPs for protein templates (targets) still require lengthy optimisation and characterisation using different ratios of monomers in order to control their size, binding affinity and specificity. In this work we successfully demonstrated that differential scanning fluorimetry (DSF) can be used to rapidly determine the optimum imprinting conditions and monomer composition required for MIP-NP design and polymerisation. This is based on the stability of the protein template and shift in apparent melting points (Tm) upon interaction with different functional acrylic monomers. The method allows for the characterisation of molecularly imprinted nanoparticles (MIP-NPs) due to the observed differences in melting point profiles between, protein-MIP-NPs complexes, pre-polymerisation mixtures and non-imprinted nanoparticles (NIP-NPs) without the need for prior purification. The technique is simple, rapid and can be carried out on most quantitative polymerase chain reaction (qPCR) thermal cyclers which have the required filters for SYPRO

Ultrasensitive detection of endotoxins using computationally designed nanoMIPs.

Novel molecularly imprinted polymer nanoparticles (nanoMIPs) were designed for endotoxin from Escherichia coli 0111:B4, using computational modeling. The screening process based on binding energy between endotoxin and each monomer was performed with 21 commonly used monomers, resulting in the selection of itaconic acid, methacrylic acid and acrylamide as functional monomers due to their strong binding interaction with the endotoxin template. The nanoMIPs were successfully synthesized with functional groups on the outer surface to aid in the immobilization onto sensor surface. The solid phase photopolymerization approach used for the synthesis of nanoMIPs ranging from 200 to 235 nm in diameter. The limit of detection and KD were significantly improved when endotoxin samples were prepared using a novel triethylamine method. This improved the efficiency of gold nanoparticle functionalization by targeting the subunits of the endotoxin. Compared to the vancomycin MIP control, the endotoxin MIPs displayed outstanding affinity and selectivity towards the endotoxin with KD values in the range of 4.4-5.3 × 10(-10) M, with limits of detection of 0.44 ± 0.02 ng mL(-1) as determined by surface plasmon resonance (SPR) sensor when itaconic acid was used as the functional monomer. The MIP surface can be regenerated >30 times without significant loss of binding activity making this approach highly cost effective for expensive analyte templates. The combination of molecular modeling and solid phase synthesis enabled the successful synthesis of nanoMIPs capable of recognition and ultrasensitive detection of endotoxins using the highly sensitive SPR biosensor with triethylamine method.

Sensitive detection of Campylobacter jejuni using nanoparticles enhanced QCM sensor.

A quartz crystal microbalance (QCM) sensor platform was used to develop an immunosensor for the detection of food pathogen Campylobacter jejuni. Rabbit polyclonal antibodies and commercially available mouse monoclonal antibodies against C. jejuni were investigated to construct direct, sandwich and gold-nanoparticles (AuNPs) amplified sandwich assays. The performance of the QCM immunosensor developed using sandwich assay by utilising the rabbit polyclonal antibody as the capture antibody and conjugated to AuNPs as the detection antibody gave the highest sensitivity. This sensor achieved a limit of detection (LOD) of 150 colony forming unit (CFU)mL(-1) of C. jejuni in solution. The QCM sensor showed excellent sensitivity and specificity for Campylobacter detection with low cross reactivity for other foodborne pathogens such as Salmonella Typhimurium, (7%) Listeria monocytogenes (3%) and Escherichia coli (0%). The development of this biosensor would help in the sensitive detection of Campylobacter which can result in reducing pre-enrichment steps; hence, reducing assay time. This work demonstrates the potential of this technology for the development of a rapid and sensitive detection method for C. jejuni.

SPR detection of cardiac troponin T for acute myocardial infarction.

A surface plasmon resonance (SPR) sensor developed for the rapid, sensitive and specific detection of cardiac troponin T (cTnT) in serum samples is reported in this work. An extensive optimisation of assay parameters was conducted to achieve optimal detection strategy. Both direct and sandwich immunoassay formats were investigated and optimised. The response obtained was enhanced further by the use of gold nanoparticles (AuNPs) conjugated to the anti-cTnT detection antibody. A regeneration method was developed to enable the reuse of the SPR sensor for multiple sample application. The SPR immunosensor showed good reproducibility for cTnT detection in the concentration range of 25-1000 ng mL(-1) and 5-400 ng mL(-1) for the direct and sandwich assays in buffer, respectively. The linear regression analysis was performed and R(2) value was found as 0.99 for both assays. In order to optimise the sensor for serum analysis, nonspecific binding of serum proteins was reduced through the use of additives in the dilution buffer. To achieve greater sensitivity, the performance of the cTnT immunosensor sandwich assay in human serum was evaluated using non-modified and AuNP modified detector antibodies. A detection limit (LOD) for the immunosensor in 50% serum was assessed as 5 ng mL(-1) cTnT for the standard sandwich assay and 0.5 ng mL(-1) cTnT when using AuNP conjugated detector antibodies with a linear dynamic range of 0.5-40 ng mL(-1). The dissociation constant was found as 3.28 × 10(-9) M using Langmuir binding model which indicates high affinity between cTnT and its antibody. The proposed SPR immunosensor has a promising potential to be developed for point-of-care testing for the early diagnosis of acute myocardial infarction (AMI). This method can also be used for the rapid detection of biomarkers in central nervous system diseases.

Comparative investigations for adenovirus recognition and quantification: Plastic or natural antibodies?

Comparative and comprehensive investigations for adenovirus recognition and detection were conducted using plastic and natural antibodies to compare three different strategies. The implementation of molecularly imprinted polymer (MIP) technology for specific and sensitive recognition of viruses with the combination of biosensors was reported. Plastic antibodies (MIPs nanoparticles) were produced for adenovirus by employing a novel solid phase synthesis method. MIP receptors were then characterised using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques prior to immobilisation on a surface plasmon resonance (SPR) sensor as affinity receptor for adenovirus detection. Two different templates were also imprinted as control MIPs (vancomycin-MIP and MS2-MIP). The specific recognition of adenovirus was investigated in the concentration range of 0.01-20 pM and the limit of detection was achieved as 0.02 pM. As an alternative to MIP receptors, direct and sandwich assays were developed for adenovirus quantification using natural antibodies. The detection limit of direct and sandwich assays were found as 0.3 pM and 0.008 pM, respectively. The kinetic data analyses were performed for three different adenovirus recognition methods and cross-reactivity studies were also conducted using MS2 phage as control virus and an excellent specificity was achieved with all assays types. This work confirmed the suitability of the MIPs SPR sensor for the detection of viruses.

Biosensors for waterborne viruses: Detection and removal.

Detection of waterborne viruses is important to eliminate and control their harmful effect as pathogens. Hence, the use of rapid and sensitive detection technologies is critically important as they can aid in investigating outbreaks and help in developing prevention strategies. To date range of viruses can contaminate drinking water sources, causing illnesses such as diarrhoea, pneumonia and gastroenteritis which can result in death. Due to their small size (nm) their complete removal from water can be difficult with current water treatment processes while being resistant to disinfectants. Available techniques for virus detection include filtration technologies, enzyme-linked immunosorbent assays and polymerase chain reaction. Although each technique has limitations, the use of biosensor technology with smart affinity materials and nanomaterials can show great potential in sensing viruses in water samples. This review reports on the latest technologies used for waterborne virus removal and detection with focus on rapid detection using biosensors.

Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

Cardiovascular disease detection using bio-sensing techniques.

Universally, cardiovascular disease (CVD) is recognised as the prime cause of death with estimates exceeding 20 million by 2015 due to heart disease and stroke. Facts regarding the disease, its classification and diagnosis are still lacking. Hence, understanding the issues involved in its initiation, its symptoms and early detection will reduce the high risk of sudden death associated with it. Biosensors developed to be used as rapid screening tools to detect disease biomarkers at the earliest stage and able to classify the condition are revolutionising CVD diagnosis and prognosis. Advances in interdisciplinary research areas have made biosensors faster, highly accurate, portable and environmentally friendly diagnostic devices. The recent advances in microfluidics and the advent of nanotechnology have resulted in the development of improved diagnostics through reduction of analysis time and integration of several clinical assays into a single, portable device as lab-on-a-chip (LOC). The development of such affinity based systems is a major drive of the rapidly growing nanotechnology industry which involves a multidisciplinary research effort encompassing nanofluidics, microelectronics and analytical chemistry. This review summarised the classification of CVD, the biomarkers used for its diagnosis, biosensors and their application including the latest developments in the field of heart-disease detection.

Detection of the inflammation biomarker C-reactive protein in serum samples: towards an optimal biosensor formula.

The development of an electrochemical immunosensor for the biomarker, C-reactive protein (CRP), is reported in this work. CRP has been used to assess inflammation and is also used in a multi-biomarker system as a predictive biomarker for cardiovascular disease risk. A gold-based working electrode sensor was developed, and the types of electrode printing inks and ink curing techniques were then optimized. The electrodes with the best performance parameters were then employed for the construction of an immunosensor for CRP by immobilizing anti-human CRP antibody on the working electrode surface. A sandwich enzyme-linked immunosorbent assay (ELISA) was then constructed after sample addition by using anti-human CRP antibody labelled with horseradish peroxidase (HRP). The signal was generated by the addition of a mediator/substrate system comprised of 3,3,5',5'-Tetramethylbenzidine dihydrochloride (TMB) and hydrogen peroxide (H2O2). Measurements were conducted using chronoamperometry at -200 mV against an integrated Ag/AgCl reference electrode. A CRP limit of detection (LOD) of 2.2 ng·mL(-1) was achieved in spiked serum samples, and performance agreement was obtained with reference to a commercial ELISA kit. The developed CRP immunosensor was able to detect a diagnostically relevant range of the biomarker in serum without the need for signal amplification using nanoparticles, paving the way for future development on a cardiac panel electrochemical point-of-care diagnostic device.

Real-time and sensitive detection of Salmonella Typhimurium using an automated quartz crystal microbalance (QCM) instrument with nanoparticles amplification.

The accidental contamination of Salmonella in raw and processed foods is a major problem for the food industry worldwide. At present many of the currently used methods for Salmonella detection are time and labour intensive. Therefore, rapid detection is a key to the prevention and identification of problems related to health and safety. This paper describes the application of a new quartz crystal microbalance (QCM) instrument with a microfluidic system for the rapid and real time detection of Salmonella Typhimurim. The QCMA-1 bare gold sensor chip which contain two sensing array was modified by covalently immobilising the monoclonal capture antibody on the active spot and a mouse IgG antibody on the control spot using a conventional amine coupling chemistry (EDC-NHS). The binding of the Salmonella cells onto the immobilised anti-Salmonella antibody alters the sensor frequency which was correlated to cells concentration in the buffer samples. Salmonella cells were detected using direct, sandwich, and sandwich assay with antibody conjugated gold-nanoparticles. The performance of the QCM immunosensor developed with gold-nanoparticles gave the highest sensitivity with a limit of detection (LOD) ~10-20 colony forming unit (CFU) ml(-1) compared to direct and sandwich assay (1.83 × 10(2) CFU ml(-1) and 1.01 × 10(2) CFU ml(-1), respectively). The sensor showed good sensitivity and selectivity for Salmonella in the presence of other bacteria in real food samples and helped in reducing the pre-enrichment step, hence, demonstrating the potential of this technology for the rapid and sensitive microbial analysis.