Portable Chronic Alcohol Consumption Monitor in Human Sweat through Square-Wave Voltammetry.

Chronic alcohol consumption is a significant financial and physical burden in the United States each year. Alcohol consumption monitors focus on establishing a state of intoxication, not assessing a user's health risks as a function of consumed alcohol. This work demonstrates a biosensor for a chronic alcohol consumption monitor through the electrochemical detection of ethyl glucuronide (EtG) in human sweat using square-wave voltammetry (SWV). A novel affinity assay was demonstrated in which monoclonal antibodies were chemically coabsorbed onto a gold electrode surface in parallel with thiolated charge transfer molecule. Concentration-dependent EtG binding was detected by measuring a reduction in the charge transfer of the sensor, manifesting as a current response during SWV measurement. A companion compact electronic reader was constructed, demonstrating comparable sensitivity to a conventional lab instrument. Both tools demonstrated a limit of detection of 0.1 µg/L and a linear dynamic range of 0.1-100 µg/L corresponding to the physiologically relevant range of EtG expression in human sweat. This device can address the need for a chronic alcohol consumption monitor toward establishing a user's long-term consumption habits to assess the risk of developing specific diseases and conditions associated with regular alcohol consumption, through integration with existing technologies.

Companion and Point-of-Care Sensor System for Rapid Multiplexed Detection of a Panel of Infectious Disease Markers.

A nanochannel-based electrochemical biosensor has been demonstrated for rapid and multiplexed detection of a panel of three biomarkers associated with rapid detection of sepsis. The label-free biosensor detected procalcitonin (PCT), lipoteichoic acid (LTA), and lipopolysaccharide (LPS) from human whole blood. The biosensor comprises a nanoporous nylon membrane integrated onto a microelectrode sensor platform for nanoconfinement effects. Charge perturbations due to biomarker binding are recorded as impedance changes using electrochemical impedance spectroscopy. The measured impedance change is used to quantitatively determine the concentration of the three biomarkers using antibody receptors from the tested sample. We were successful in detecting and quantifying the three biomarkers from whole blood. The limit of detection was 0.1 ng/mL for PCT and 1 µg/mL for LPS and LTA. The sensor was able to demonstrate a dynamic range of detection from 01.1 ng/mL to 10 µg/mL for PCT and from 1 µg/mL to 1000 µg/mL for LPS and LTA biomarkers. This novel technology has promising preliminary results toward the design of sensors for rapid and sensitive detection of the three panel biomarkers in whole blood toward diagnosis and classification of sepsis.

Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process.

Flexible nanoporous tunable electrical double layer biosensors for sweat diagnostics.

An ultra-sensitive and highly specific electrical double layer (EDL) modulated biosensor, using nanoporous flexible substrates for wearable diagnostics is demonstrated with the detection of the stress biomarker cortisol in synthetic and human sweat. Zinc oxide thin film was used as active region in contact with the liquid i.e. synthetic and human sweat containing the biomolecules. Cortisol detection in sweat was accomplished by measuring and quantifying impedance changes due to modulation of the double layer capacitance within the electrical double layer through the application of a low orthogonally directed alternating current (AC) electric field. The EDL formed at the liquid-semiconductor interface was amplified in the presence of the nanoporous flexible substrate allowing for measuring the changes in the alternating current impedance signal due to the antibody-hormone interactions at diagnostically relevant concentrations. High sensitivity of detection of 1 pg/mL or 2.75 pmol cortisol in synthetic sweat and 1 ng/mL in human sweat is demonstrated with these novel biosensors. Specificity in synthetic sweat was demonstrated using a cytokine IL-1ß. Cortisol detection in human sweat was demonstrated over a concentration range from 10-200 ng/mL.

Electrical nanowell diagnostics sensors for rapid and ultrasensitive detection of prostate-specific antigen.

AIM: Alumina nanowell based disposable diagnostic biosensor for detecting and quantifying levels of prostate-specific antigen (PSA) from human serum has been designed, fabricated and tested. MATERIALS & METHODS: The biosensors were designed by integrating nanoporous alumina membranes onto printed circuit board platforms, resulting in the generation of high-density nanowell arrays with gold base electrodes. The size and density of the nanowells were leveraged toward achieving sieving action for size-based exclusion of nonspecific molecules and size-based confinement of the target PSA molecules. RESULTS & CONCLUSION: We demonstrated PSA detection between 0.01 and 1000 ng/ml and detection and quantification of PSA from a 17 patient cohort validated using the Beckman Access system with >95% correlation.

Cellular level classification of breast cancer through proteomic markers using nanochannel array sensors.

AIMS: A nanochannel-based sensor for cellular level classification of breast cancer metastasis has been designed. Our approach to the classification of cell's likelihood to metastasize is based on screening for levels of expression of specific proteomic biomarkers associated with breast cancer stem cells. MATERIALS & METHODS: Proteomic activity for four breast cancer cell lines for three specific markers (PDGFR, ALDH1A1 and ALDH1A3) was quantified. The nanochannel sensor is an electrochemical immunoassay and comprises of alumina nanochannel arrays integrated on to a gold microelectronic platform. The sensor operates on the principle of electrochemical impedance spectroscopy. RESULTS & CONCLUSION: Test cell lysate samples from SUM159 invasive, SUM159 noninvasive, HC1143 and DCIS cell lines were classified as those having a high likelihood of metastases based on the levels of proteomic activity evaluated against the three key markers. The lowest proteomic activity measured was 0.1 ng/ml with PDGFR, 100 ng/ml with ALDH1A1 and 100 ng/ml with ALDH1A3, correlating to the detection of unit stem cell count. Original submitted 9 November 2012; Revised submitted 29 July 2013.

Antibody-conjugated gold nanoparticle-based immunosensor for ultra-sensitive detection of troponin-T.

The technology presented in this study demonstrates the feasibility of integrating nanostructures onto the surface of an electrical platform to achieve enhanced detection of the cardiac biomarker, troponin-T. A polymer microcontact printing technique was modified using printed circuit boards as molds for the application of gold nanoparticles onto microelectrode-patterned glass substrates. The microelectrodes were designed to support electrical impedance spectroscopy measurements and fabricated using standard photolithography methods. Capture antibodies specific to troponin-T were functionalized onto the surface of gold nanoparticles by using a thiol-based cross-linking molecule. The antibody-conjugated gold nanoparticles were stamped onto the electrodes using a matching pattern imprinted onto an elastomeric mold. As a control to validate the efficacy of the nanotextured surface on the glass substrate, an electroplated printed circuit board was also used. The incorporation of gold nanoparticles showed significant amplification of the electro-ionic signals generated through binding of the antigen to its capture antibody. Enhanced sensitivity was demonstrated through detection of the target biomarker in the femtogram per milliliter range in buffer solution and biological media. In the absence of gold nanoparticles, the sensor demonstrated detection of troponin-T at higher concentration points. This study illustrates a robust method for developing a more sensitive, label-free biosensor.

Ultra-sensitive electrical immunoassay biosensors using nanotextured zinc oxide thin films on printed circuit board platforms.

This study demonstrates the development of nanotextured zinc oxide (ZnO) thin films sputter deposited on printed circuit boards (PCB) to enhance the capability in detecting low concentrations of the protein troponin-T. The presence of this particular biomarker in the bloodstream is a direct indicator of current and/or future risk of various forms of cardiovascular diseases. Electrical transduction through impedance spectroscopy was used to detect troponin-T functionalized immunoassays on nanotextured ZnO surfaces. Calibration of the immunoassay was performed by measuring the impedance changes resulting from the binding of increasing concentrations of troponin-T to the immobilized antibodies on the ZnO surface in (i) phosphate buffered saline (PBS) and (ii) human serum. The limit of detection achieved using this platform was 10 fg/mL and 100 fg/mL in PBS and human serum, respectively. Enhanced detection of troponin-T was found to correlate to the oxygen vacancies in the ZnO thin film. PCB was chosen as the substrate for ease of integration with microelectronic device manufacturing.

Nanosensor electrical immunoassay for quantitative detection of NT-pro brain natriuretic peptide.

AIM: To demonstrate a label-free electrical immunoassay for profiling vascular biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP) associated with improved cardiac risk prediction. MATERIALS & METHODS: A high-density nanowell-based electrical immunoassay has been designed by integrating nanoporous aluminum oxide onto printed circuit board chips for the detection of NT-proBNP. The concentration of the biomarker is quantitatively determined by measuring impedance changes to the electrical double layer within the nanowells using electrochemical impedance spectroscopy. Detection sensitivity in the fg/ml range was obtained due to spatial confinement of the target biomarkers in size-matched nanowells. RESULTS & DISCUSSION: Electrical immunoassay performance was determined for the detection of NT-proBNP in phosphate-buffered saline (PBS) and human serum (HS). The lower limit of detection for the sensor was observed to be 10 fg/ml in PBS and 500 fg/ml in HS. The upper limit of detection was observed to be 500 fg/ml in PBS and 500 ng/ml in HS. CONCLUSION: A label-free technique for detection of NT-proBNP at clinically relevant concentrations for evaluating cardiac risk is demonstrated. High sensitivity and specificity, robust detection and low volume (100 µl) per assay project the technology to be a successful competitor to traditional ELISA-based techniques.

Design of a high sensitive non-faradaic impedimetric sensor.

An electrochemical approach towards identifying antigen-antibody binding interactions is studied by using a non-faradaic impedimetric sensor fabricated on a printed circuit board (PCB) chip. An electrical methodology for detecting protein interactions at ultra-low concentrations (in the femtogram/mL) regime has been demonstrated. Nanoporous alumina with pore diameter of 200nm and pore depth of 250 nm was used as the signal amplifying medium. Cardiac biomarker, brain natriuretic peptide (BNP) was used as the study marker in characterizing the sensor's sensitivity. A sensitivity of 10 femtogram/mL was determined based on the impedimetric signal response. Sensitivity was determined through Nyquist plot analysis for the non-faradaic interactions of the protein biomolecules. This paper is the first demonstration of clinically relevant limit of detection with the BNP biomarker.