Impedance Biosensor Research Articles

Abstract P3-10-20: Sub-group of HER2- breast cancer patients with hyperactive and co-involved c-Met and ErbB pathways identified: Functional signal profiling test identifies patient group that may benefit from c-Met and pan-HER combination therapy

Abstract Background: Biological factors other than c-Met status, such as c-Met and ErbB signaling activity, may be important to measure when identifying patients eligible for c-Met therapies. A new assay using an impedance biosensor was developed to measure c-Met and ErbB signaling activity of live tumor cells. The CELx Multi-Pathway Signaling Function (CELx MP) Test measures an individual patient's ex vivo live tumor cell response in real-time to specific ErbB and c-Met agonists and antagonists to diagnose breast tumors with hyperactive HER1, HER2, HER3, and c-MET signaling. This study set out to: 1) determine the prevalence of hyperactive c-Met and ErbB family signaling amongst HER2- breast cancer patients; and 2) characterize potential cross-talk between c-Met and ErbB pathways. Methods: For the prevalence study, fresh breast tumor specimens were obtained from 74 HER2- breast cancer patients. The amount of HER1, HER2, HER3, and c-Met for each specimen was determined using FACS. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) alone and in combination, with or without ErbB and c-Met antagonists (2C4, a HER2 mAb dimerization inhibitor, tepotinib, a c-Met TKI, or neratinib, a pan-HER TKI) was measured using an xCELLigence RTCA impedance biosensor. From these responses, HER1, HER3, and c-Met signaling initiated by their respective agonists was quantified. The net amount of HER2 participation in EGF and NRG signaling was also quantified. Signaling activity above a previously determined cutpoint was used to identify abnormal levels of HER1, HER2, HER3 and c-Met signaling activity. For the cross-talk study, three primary HER2- breast cancer specimens with hyperactive c-Met and ErbB signaling were obtained. Response to HGF, EGF, and NRG1 alone, with or without tepotinib, was measured for these specimens using an impedance biosensor. Results: The FACS analysis found all 74 tumor samples had normally expressed amounts of HER1, HER2, HER3, and c-Met. Of these samples, 20 of 74 (27.0%; 95% CI=18%-38%) had hyperactive c-Met signaling coincident with hyperactive signaling from at least one ErbB pathway. In each patient sample, neratinib combined with tepotinib inhibited virtually all signaling activity initiated with a combination EGF, NRG1 and HGF. In the cross-talk analysis of the three tumor samples, signaling response to EGF or NRG1 when combined with a c-Met antagonist was 9%-98% higher than signaling response measured with EGF or NRG1 alone. Conclusions: This test found a significant sub-set of HER2- breast cancer patients with coincidental hyperactive c-Met and ErbB signaling tumors that respond ex vivo to a combination of pan-HER and c-Met TKI's. The unexpected increase in EGF and NRG1 signaling in the presence of a c-Met antagonist provides strong evidence that c-Met and ErbB signaling is co-involved and may explain why a c-Met TKI is not an effective antagonistic when c-Met is hyperactive for this patient sub-set. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Sub-group of HER2- breast cancer patients with hyperactive and co-involved c-Met and ErbB pathways identified: Functional signal profiling test identifies patient group that may benefit from c-Met and pan-HER combination therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-20.

Abstract P2-05-05: Evaluation of pan-HER and c-MET inhibitors tested ex vivo in primary HER2- breast cancer cells with hyperactive c-MET and ErbB family signaling

Abstract Background: To elucidate the role of c-Met signaling and its involvement with ErbB signaling as a cancer driver, a new assay using an impedance biosensor, the CELx multi-pathway signaling function (CELx MP) test, was developed. The CELx MP Test measures ex vivo real-time live cell response to specific ErbB and c-Met agonists to diagnose breast tumors with hyperactive HER1, HER2, HER3, HER4, and c-MET signaling activity. A recent study quantified c-MET and ErbB-driven signaling activity in epithelial cell samples derived from fresh breast tumor specimens obtained from 74 HER2- breast cancer patients. Of the cell samples tested, 20 of 74, (27.0%; 95% CI=18%-38%) had both hyperactive c-MET signaling and at least one hyperactive ErbB-family receptor signaling. Using primary breast cancer cells with hyperactive c-MET and ErbB signaling and the CELx MP test, the current study set out to: 1) determine the IC50 values of six pan-HER and five c-MET inhibitors; and 2) characterize the efficacy of combinations of each pan-HER inhibitor with each c-MET inhibitor. Methods: Epithelial cells from six HER2- tumor specimens with hyperactive c-MET and ErbB-driven signaling were obtained. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) alone and in combination, with or without one of six pan-HER antagonists (neratinib, lapatinib, ibrutinib, dacomitinib, sapitinib, poziotinib) or one of five c-MET antagonists (tepotinib, cabozantinib, crizotinib, capmatinib, or savolitinib) was quantified using an xCELLigence RTCA impedance biosensor. Each individual drug IC50 was determined using a 1000-fold, 5-point, dose response curve with a single fixed concentration of a corresponding agonist. For the drug combination efficacy studies, fixed concentrations of the agonist mixture and clinically relevant concentrations of combinations of the antagonists were used to determine the percentage inhibition of the ErbB and c-MET signaling. Results: The IC50 values for the individual c-MET and pan-HER inhibitors ranged from 3.10nM - 28nM and 2.67nM – 137.27nM, respectively. In the drug efficacy studies, an average of at least 80% of the ErbB and c-MET signaling activated by NRG1, EGF, and HGF co-stimulation was inhibited by each combination of c-MET and pan-HER inhibitors. c-Met Inhibitor Resultsc-MET inhibitorIC50 (nM)Avg Inhibition (%) w/diff ErbBi's Capmatinib3.1094Savolitinib3.3298Tepotinib14.7096Cabozantinib27.3699Crizotinib28.21100 Pan-HER Inhibitor ResultsPan-HER inhibitorIC50 (nM)Avg Inhibition (%) w/diff c-METi's Poziotinib2.67100Neratinib4.81100Ibrutinib13.1099Dacomitinib22.06100Sapitinib41.2898Lapatinib137.2780 Conclusions: The CELx MP test using live cells measures IC50 values comparable to those derived using cell-free methods. Every combination of pan-HER and c-MET inhibitors provided comparably high (at least 80%) levels of inhibitory effect ex vivo. This suggests the sub-group of HER2- breast cancer patients diagnosed with coincident hyperactive c-MET and ErbB signaling by the CELx Test may respond to virtually any pan-HER and c-Met inhibitor combination. Studying combinations designed to minimize drug toxicities without sacrificing efficacy should thus be possible. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Evaluation of pan-HER and c-MET inhibitors tested ex vivo in primary HER2- breast cancer cells with hyperactive c-MET and ErbB family signaling [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-05-05.

Abstract P3-10-15: Evaluating contribution of hyperactive c-Met and ErbB signaling to tumor progression in mouse breast tumor xenografts: An in vivo study of c-Met and ErbB targeted therapies

Abstract Background: Hyperactive c-Met signaling, including cross-talk between c-Met and ErbB family receptors, is suspected of contributing to tumor progression in a variety of cancer types. Clinical trials evaluating c-Met inhibitors alone and in combination with other targeted therapies, have produced mostly negative results in patients with amplified c-Met. This suggests other biological factors, such as c-Met and ErbB signaling activity, may be important to measure when identifying patients eligible for c-Met therapies. To measure the c-Met and ErbB signaling activity of a patient's live tumor cells, a new assay using an impedance biosensor, the CELx multi-pathway signaling function (CELx MP) test, was developed. The CELx MP test measures a patient's ex vivo live tumor cell response in real-time to specific ErbB and c-Met agonists to diagnose breast cancer tumors with hyperactive HER1, HER2, HER3, HER4, and c-MET signaling activity. In this study, to further elucidate the role of c-Met signaling and its potential involvement with ErbB signaling as a cancer driver, we studied in vivo response to a c-Met inhibitor (tepotinib), an EGFR inhibitor (erlotinib), a pan-HER inhibitor (neratinib), and a combination of these therapies using breast tumor xenograft models. Methods: HCC1954, a HER2+ cell line with hyperactive c-Met and EGFR signaling and normal HER3 and HER2-driven signaling, according to the CELx MP test, was studied. Sixty 4-5-week-old female NSG mice were injected with two million cells. Mice were randomly assigned to either a control group that received Captisol or one of five treatment groups that received either neratinib, tepotinib, erlotinib, erlotinib and tepotinib, or neratinib and tepotinib for 17 days. Results: The most effective treatment was the combination of neratinib plus tepotinib, where the average tumor size reduction relative to the control group was 71% (p=0.0003). The average tumor size in the neratinib plus tepotinib treated group was 37% smaller (p=0.049) than the neratinib treated group, and 67% smaller (p=0.0026) than the tepotinib treated group. In the erlotinib plus tepotinib treatment group, the average tumor size was 51% smaller than the control group tumors, but the difference was not statistically significant (p=0.11). No significant difference in tumor size was found between the control group and the erlotinib or tepotinib treated groups. Conclusions: The results demonstrate that hyperactive and coincident c-Met and EGFR signaling contributes to the progression of certain breast cancers. This breast cancer sub-type is more responsive to treatment with a c-Met inhibitor plus a pan-HER inhibitor than a c-Met inhibitor plus an EGFR inhibitor or any of the single agents studied. This suggests that when hyperactive c-Met signaling and a hyperactive ErbB family member is present in a patient's tumor, each of the ErbB pathways as well as the c-Met pathway must be inhibited to treat the tumor most effectively. These findings provide strong evidence that HER2-negative breast cancer patients with coincident hyperactive c-MET and ErbB signaling may respond to treatment with a combination of pan-HER and c-Met inhibitors. Citation Format: Laing LG, Burns DJ, Khan S, MacNeil IA, Rich BE, Kharbush SM, Soltani SM, Sullivan BF. Evaluating contribution of hyperactive c-Met and ErbB signaling to tumor progression in mouse breast tumor xenografts: An in vivo study of c-Met and ErbB targeted therapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-15.

Microfluidic based impedance biosensor for pathogens detection in food products.

A MEMS-based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonellatyphimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria.

Effect of the nonspecific binding in differential impedance biosensing.

The detection limits of impedance biosensors are dictated by the presence of background nonspecific binding, yet almost all the detection limits reported in the literature are determined using a clean buffer solution without confronting this real challenge. In this work, the authors employed the simplest "differential" impedance biosensor, composed of poly-l-lysine-polyethylene glycol-biotin-coated gold electrodes for the detection of streptavidin in the presence of 0.1% fetal calf serum, and studied the effect of the nonspecific binding on the performance of the differential impedance biosensing. The lowest streptavidin concentration detected by the system (5 μg/ml) was 1 order of magnitude higher (worse) than that from a previously demonstrated impedance biosensor where avidin was detected in the absence of background proteins. Interestingly, the origin of the differential signal was not due to the electrochemical properties of streptavidin itself but was that of the serum, where the coverage of the electrode by streptavidin indirectly modulated the electrical signal by suppressing the accessibility of the serum to the electrode.

Electrochemical Impedance Biosensor for the Determination of Lipopolysaccharide Using Peptide as the Recognition Molecule

A simple and sensitive electrochemical impedance spectroscopy (EIS) biosensor was designed for the determination of lipopolysaccharide (LPS). The gold nanoparticles (GNPs) were electrochemically deposited on the glassy carbon electrode as a base electrode and the biosensor was fabricated by self-assembling a special peptide (GIGKLHSAGKF) used as molecular recognition onto the GNPs surface according to the Au–S bond. Polyethylene glycol and 6-mercapto-1-hexanol were used to block the remaining sites of the modified electrode surface. When binding of LPS, the biosensor had a good response to different concentrations of LPS and the charge transfer resistance of the biosensor was directly related to the logarithm of LPS concentrations in the range of 0.01 pg mL-1-1.0 ng mL-1 with a limit of detection of 2.0 fg mL-1. The characteristics of the electrochemically deposited GNPs and the optimization concentration and the self-assembly time of peptide, as well as the performance of the EIS biosensors for LPS were investigated. The biosensor was used for real serum sample determination with satisfactory results.

Low concentration E. coli O157:H7 bacteria sensing using microfluidic MEMS biosensor.

This paper reports the design, fabrication, and testing of a microfluidic MEMS biosensor for rapid sensing of low concentration Escherichia coli O157:H7. It consists of a specially designed focusing and sensing region, which enables the biosensor to detect low concentration of bacterial cells. The focusing region consists of a ramped vertical electrode pair made of electroplated gold along with tilted thin film finger pairs (45°) embedded inside a microchannel. The focusing region generates positive dielectrophoresis force, which moves the cells towards the edges of the tilted thin film electrode fingers, located at the center of the microchannel. The fluidic drag force then carries the focused cells to the sensing region, where three interdigitated electrode arrays (IDEAs) with 30, 20, and 10 pairs, respectively, are embedded inside the microchannel. This technique resulted in highly concentrated samples in the sensing region. The sensing IDEAs are functionalized with the anti-E. coli antibody for specific sensing of E. coli 0157:H7. As E. coli binds to the antibody, it results in an impedance change, which is measured across a wide frequency range of 100 Hz-10 MHz. The biosensor was fabricated on a glass substrate using the SU8 epoxy resist to form the microchannel, gold electroplating to form the vertical focusing electrode pair, a thin gold film to form the sensing electrode, the finger electrodes, traces and bonding pads, and polydimethylsiloxane to seal the device. The microfluidic impedance biosensor was tested with various low concentration bacterial samples and was able to detect bacterial concentration, as low as 39 CFU/ml with a total sensing time of 2 h.

An impedance biosensor for simultaneous detection of low concentration of Salmonella serogroups in poultry and fresh produce samples

This paper reports the design, fabrication and testing of a microfluidic based impedance biosensor for rapid and simultaneous detection of three Salmonella serogroups. The microfluidic device consists of three microchannels, each one includes a region for focusing the Salmonella cells into the centerline of the microchannel and direct them toward the sensing region to obtain highly concentrated samples using positive dielectrophoresis force. A region for bacteria sensing consists of interdigitated electrode (IDE) array with 10 pairs of fingers. Three types of Salmonella antibodies (type B, D and E) were mixed separately with the cross-linker (Sulfo-LC-SPDP) to enhance the immobalization of the antibodies to the detection electrodes. The electrode surfaces was then functionalized with the three mixtures, one for each channel. As target antigen binds to the antibody, it results in impedance change. The Salmonella samples were spiked with Salmonella type B, introduced into the biosensor via the sample inlet into the focusing region, and then toward the sensing region where they bind to the immobilized antibody, causing a change in the impedance. The performance of the devices was tested using single Salmonella serotype B and two Salmonella serotypes B, and D, with a limit of detection of 7 cells/ml. The biosensor was also able to differentiate live from dead bacteria eliminating the false positive results. Finally, the device was also able to detect Salmonella selectively when other type of pathogen was present.

An integrated impedance biosensor platform for detection of pathogens in poultry products

This paper presents an impedance-based biosensor for rapid and simultaneous detection of Salmonella serotypes B, D, and E with very low concentration. The biosensor consists of a focusing region, and three detection regions. The cells focusing was achieved using a ramp down electroplated vertical electrode pair along with tilted thin film finger pairs that generate p-DEP forces to focus and concentrate the bacterial cells into the center of the microchannel, and direct them toward the detection region. The detection regions consist of three interdigitated electrode arrays (IDEA), each with 20 pairs of finger coated with a mixture of anti-Salmonella antibody and crosslinker to enhance the adhesion to IDEA. The impedance changes as the target Salmonella binds to the antibody. The biosensor has showed excellent performance as proven by the detection of a single Salmonella serotype B, and simultaneous detection of two Salmonella serotypes B and D with a limit of detection (LOD) of 8 Cells/ml in ready-to-eat turkey samples, the addition of focusing capability improved the measured signal by a factor of between 4–4.5, the total detection time of 45 minutes, selectivity of the sensor on different types of bacterial cells, and the ability to distinguish between dead and live cells.

Impedance Detection of 3‐Phenoxybenzoic Acid Comparing Wholes Antibodies and Antibody Fragments for Biomolecular Recognition

AbstractAntibody fragments for detection of 3‐phenoxybenzoic acid (3‐PBA) are created by disulfide bond cleavage with tris(2‐carboxyethyl)phosphine (TCEP). These antibody fragments are employed to improve the sensitivity for 3‐PBA detection by electrochemical impedance spectroscopy (EIS). The sensitivity and detection limit are compared for biomolecular recognition by both polyclonal antibodies and antibody fragments immobilized through amide bond formation atop a self‐assembled monolayer (SAM) on an Au electrode, as well as antibody fragments directly attached to Au through Au−S covalent bond formation. The detection limit for 3‐PBA is dramatically improved (1.1×10−8 M) for direct immobilization of antibody fragments through Au−S covalent bond formation relative to immobilization of either polyclonal antibodies (2.1×10−5 M) or antibody fragments (1.7×10−5 M) through amide bond formation. This can be attributed primarily to the much lower charge transfer resistance (Rct) and higher ionic permeability obtained at biosensor interfaces that do not include an Au‐thiol SAM, allowing easier detection of further changes in Rct. In addition, the exponential dependence of the electron tunnelling rate on biomolecular film thickness increases the sensitivity of the antibody fragments modestly relative to polyclonal antibodies. This is the first quantitative comparison of the use of antibodies and antibody fragments for biomolecular recognition within impedance biosensors.

Selection of ssDNA aptamers for the development of impedance biosensor to detect sarcosine

This chapter provides an outline of the importance of microfluidic-based biosensors for detection of biomolecules. With an introduction of microfluidic biosensors, we discuss fundamentals of microfluidics along with their potential merits and demerits. A historical background of microfluidic devices with its current research activities has also been elucidated. A brief description relating to the suitable materials used for microfluidic fabrication has been given. In particular, utilization of nanostructured materials for integration with microfluidic assembly has been described. Besides this, efforts have been made to discuss the suitable detection strategies using microfluidic assembly to improve biosensor efficacy in terms of sensitivity, selectivity, and stability compared with that of other methods.

Analysis of Cytotoxic Effects of Chemotherapeutic Agents for Gastrointestinal Cancer with Cell-based Impedance Biosensor

Analysis of Cytotoxic Effects of Chemotherapeutic Agents for Gastrointestinal Cancer with Cell-based Impedance Biosensor

Ultrasensitive Electrochemical Detection of Clostridium perfringens DNA Based Morphology-Dependent DNA Adsorption Properties of CeO₂ Nanorods in Dairy Products.

Foodborne pathogens such as Clostridium perfringens can cause diverse illnesses and seriously threaten to human health, yet far less attention has been given to detecting these pathogenic bacteria. Herein, two morphologies of nanoceria were synthesized via adjusting the concentration of NaOH, and CeO2 nanorod has been utilized as sensing material to achieve sensitive and selective detection of C. perfringens DNA sequence due to its strong adsorption ability towards DNA compared to nanoparticle. The DNA probe was tightly immobilized on CeO2/chitosan modified electrode surface via metal coordination, and the DNA surface density was 2.51 × 10−10 mol/cm2. Under optimal experimental conditions, the electrochemical impedance biosensor displays favorable selectivity toward target DNA in comparison with base-mismatched and non-complementary DNA. The dynamic linear range of the proposed biosensor for detecting oligonucleotide sequence of Clostridium perfringens was from 1.0 × 10−14 to 1.0 × 10−7 mol/L. The detection limit was 7.06 × 10−15 mol/L. In comparison, differential pulse voltammetry (DPV) method quantified the target DNA with a detection limit of 1.95 × 10−15 mol/L. Moreover, the DNA biosensor could detect C. perfringens extracted DNA in dairy products and provided a potential application in food quality control.

Tracing the pH dependent activation of autophagy in cancer cells by silicon nanowire-based impedance biosensor

Monitoring the pH dependent behavior of normal and cancer cells by impedimetric biosensor based on Silicon Nanowires (SiNWs) was introduced to diagnose the invasive cancer cells. Autophagy as a biologically activated process in invasive cancer cells during acidosis, protect them from apoptosis in lower pH which presented in our work. As the autophagy is the only activated pathways which can maintain cellular proliferation in acidic media, responses of SiNW-ECIS in acidified cells could be correlated to the probability of autophagy activation in normal or cancer cells. In contrast, cell survival pathway wasn’t activated in low-grade cancer cells which resulted in their acidosis. The measured electrical resistance of MCF10, MCF7, and MDA-MB468 cell lines, by SiNW sensor, in normal and acidic media were matched by the biological analyses of their vital functions. Invasive cancer cells exhibited increased electrical resistance in pH 6.5 meanwhile the two other types of the breast cells exhibited sharp (MCF10) and moderate (MCF7) decrease in their resistance. This procedure would be a new trend in microenvironment based cancer investigation.

Abstract P2-09-22: Use of a functional signal profiling test with high sensitivity and specificity to determine the prevalence of abnormal HER2-driven signaling activity in the HER2-negative breast cancer patient population: New patient group may benefit from HER2 therapy

Abstract Background: Biological factors, such as HER2 signaling activity, may be important to measure in addition to expression and amplification of HER2 when identifying patients eligible for HER2 therapies. The CELx HER2 Signaling Function (CELx HSF) Test measures HER2 signaling activity in live tumor cells using a label-free impedance biosensor to identify HER2-negative breast cancer patients likely to be responsive to treatment with anti-HER2 therapies. Previous studies quantified HER2-driven signaling activity in a training set (N=34) of primary tissue samples from HER2-negative breast cancer patients and found 21% of the samples had abnormal HER2 signaling. Other studies confirmed that anti-HER2 therapies, such as trastuzumab, pertuzumab, afatinib, and neratinib, are as effective in inhibiting HER2-driven signaling activity in HER2- tumor cells as they are in HER2+ tumor cells. This study set out to confirm the prevalence of abnormal HER2 signaling amongst HER2-negative breast cancer patients in a larger sample (N=114) and to characterize the sensitivity and specificity of the CELx HSF Test. Methods: A validation set of de-identified fresh breast tumor specimens were obtained from 114 HER2- breast cancer patients. Real time live cell response to specific HER2 agonists (NRG1b or EGF) with or without an antagonist (HER2 dimerization inhibitor) was measured using an impedance biosensor. From these responses, the net amount of HER2 participation in HER2 signaling initiated by the HER2 agonists was quantified. Samples with HER2 signaling activity levels above a previously determined cut-off value were identified as abnormal. Results: Of the HER2- breast tumor cell samples tested, 27 of 114 patients (23.7%; 95% CI=17%-32%) had abnormal HER2 signaling activity. Little or no correlation was found between a patient's HER2 signaling activity and their estrogen receptor status or tumor grade. To compare the results obtained from the training set of 34 patients and the current set of 114 patients, the Kolmogorov-Smirnov two-sample test was applied (D=0.17, P-value 0.45) and found no significant difference between the training and validation sets. A normal mixture model was fitted to the new 114 patient data set and found that HER2- breast cancer patients fall into three distinct groups (abnormal, normal, low). Patients falling into the abnormal group had mean HER2 signaling scores 4.5 standard deviations above the mean score of the normal group. A ROC curve constructed with this data projects that both the sensitivity and specificity of the CELx HSF Test would be greater than 90%. Conclusions: These results confirm that a clinically relevant proportion of HER2- breast cancer patients, approximately 20%, have tumors with abnormal HER2-signaling activity and may benefit from HER2 therapy. With high specificity and sensitivity, the CELx HSF test may be suitable as a companion diagnostic to identify new patients eligible to receive HER2 therapies. An interventional trial to evaluate the efficacy of trastuzumab and pertuzumab in HER2- patients selected with the CELx HSF test is underway. Citation Format: Laing LG, Burns DJ, MacNeil IA, Rich BE, Myhre S, Soltani S, Sullivan BF. Use of a functional signal profiling test with high sensitivity and specificity to determine the prevalence of abnormal HER2-driven signaling activity in the HER2-negative breast cancer patient population: New patient group may benefit from HER2 therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-22.

A microfluidic impedance biosensor based on immunomagnetic separation and urease catalysis for continuous-flow detection of E. coli O157:H7

Early screening of foodborne pathogens is a key to control the outbreaks of foodborne illnesses. In this study, a microfluidic impedance biosensor combined with the immune magnetic nanoparticles (MNPs) for bacteria separation, the urease for biological signal amplification and the microfluidic chip for impedance measurement was developed for rapid, sensitive and continuous-flow detection of E. coli O157:H7. After the streptavidin modified MNPs were conjugated with the biotinylated polyclonal antibodies (PAbs) to form the immune MNPs, the target bacteria was first separated by the MNPs from the background to form the MNP-bacteria complexes. Then, the gold nanoparticles (GNPs) modified with the urease and the aptamers against E. coli O157:H7 were conjugated with the MNP-bacteria to form the MNP-bacteria-GNP-urease complexes. Finally, the complexes were used to catalyze the hydrolysis of urea into ammonium carbonate, leading to the decrease in the impedance. The impedance was online measured by this proposed biosensor and analyzed using the impedance normalization to determine the concentration of E. coli O157:H7. A good linear relationship between the impedance relative change rate of the catalysate and the concentration of the bacteria was obtained with low detection limit of 12 CFU/mL.

Modeling and development of screen-printed impedance biosensor for cytotoxicity studies of lung carcinoma cells.

Electrical cell-substrate impedance sensing (ECIS) is a powerful technique to monitor real-time cell behavior. In this study, an ECIS biosensor formed using two interdigitated electrode structures (IDEs) was used to monitor cell behavior and its response to toxicants. Three different sensors with varied electrode spacing were first modeled using COMSOL Multiphysics and then fabricated and tested. The silver/silver chloride IDEs were fabricated using a screen-printing technique and incorporated with polydimethylsiloxane (PDMS) cell culture wells. To study the effectiveness of the biosensor, A549 lung carcinoma cells were seeded in the culture wells together with collagen as an extracellular matrix (ECM) to promote cell attachment on electrodes. A549 cells were cultured in the chambers and impedance measurements were taken at 12-h intervals for 120h. Cell index (CI) for both designs were calculated from the impedance measurement and plotted in comparison with the growth profile of the cells in T-flasks. To verify that the ECIS biosensor can also be used to study cell response to toxicants, the A549 cells were also treated with anti-cancer drug, paclitaxel, and its responses were monitored over 5days. Both simulation and experimental results show better sensitivity for smaller spacing between electrodes. Graphical abstract The fabricated impedance biosensor used screen-printed silver/silver chloride IDEs. Simulation and experimental results show better sensitivity for smaller between electrodes.

Label-free impedimetric glycan biosensor for quantitative evaluation interactions between pathogenic bacteria and mannose

In order to understanding the pathogenic mechanism of infectious diseases, it was important to study the selective recognition and interaction between pathogenic bacteria and host cells. In this paper, a novel electrochemical impedance biosensor was proposed, in which the Man/MUA-MH/Au sensing surface (Man: mannose; MUA: 11-mercapto eleven acid; MH: 6-mercapto hexanol) was fabricated and was of good biologically active and stability. The capture capacity of the designed sensing surface for S. typhimurium ATCC14028, E. coli JM109 and E. coli DH5α were characterized by Electrochemical impedance spectroscopy (EIS). According to Randless equivalent circuit and the Frumkin isotherm model, electron transfer impedance (Ret) was obtained and the binding affinity of the three bacteria and Man was calculated. It was shown that the sensing surface had a better binding affinity for S. typhimurium ATCC14028 with KADS(S.T.) = 2.16 × 106 CFU/mL. The impedance normalized value NIC (S.T.-Man) was of a good linear relationship with the logarithm of bacterial concentration (R2 = 0.96) in the range of 50–1000 CFU/mL. The detection limit was 50 CFU/mL. Meanwhile, the E. coli JM109 which expresses type 1 fimbriae was also adsorbed on the designed sensing surface with KADS(JM109) = 5.84 × 103 CFU/mL. It was illustrated that the novel electrochemical impedance biosensor could be more rapid and reliable for studying interactions between pathogen and glycan, and it was also perspective for a new point-of-care diagnostic tool for evaluating the pathogenicity bacteria.

Content Cytotoxicity Studies of Colorectal Carcinoma Cells Using Printed Impedance Sensors

Monitoring the effectiveness of drugs on cancer cells is crucial for chemotherapeutics studies. In-vitro cell-based biosensors can be used as an alternative for characteristic studies of cells’ response to drugs. Cell-based sensors provide real-time measurements and require smaller sample volumes compared to conventional T-flask measurement methods. This paper presents a biosensor that detects in real-time, impedance variations of human colon cancer, HCT-116 cells when treated with anti-cancer agent, 5-Fluorouracil (5-FU). Two different extracellular matrix (ECM); polyaniline and gelatin were tested and evaluated in terms of attachment quality. Polyaniline was found to provide the best attachment for HCT-116 cells and was used for cytotoxicity studies. Cytokinetic behavior indicated that 5-FU inhibited HCT-116 cells at IC50 of 6.8 µg/mL. Trypan blue exclusion method for testing cell viability was used to validate the impedance measurements, where the cancer cell concentrations were reduced to ~35% when treated with 2.5 µg/mL, and 50% when treated with 6.8 µg/mL. The results generated by the microfabricated impedance biosensor are comparable to the Trypan blue method since both gave similar cell growth trend. It can be concluded that the impedance biosensor has potential to be used as an alternative method in drug testing applications.

A DNAzyme-based Electrochemical Impedance Biosensor for Highly Sensitive Detection of Cu2+ Ions in Aqueous Solution

A DNAzyme-based Electrochemical Impedance Biosensor for Highly Sensitive Detection of Cu2+ Ions in Aqueous Solution