Modeling pulmonary cystic fibrosis in a human lung airway-on-a-chip.

BACKGROUND: Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which results in impaired airway mucociliary clearance, inflammation, infection, and respiratory insufficiency. The development of new therapeutics for CF are limited by the lack of reliable preclinical models that recapitulate the structural, immunological, and bioelectrical features of human CF lungs. METHODS: We leveraged organ-on-a-chip technology to develop a microfluidic device lined by primary human CF bronchial epithelial cells grown under an air-liquid interface and interfaced with pulmonary microvascular endothelial cells (CF Airway Chip) exposed to fluid flow. The responses of CF and healthy Airway Chips were analyzed in the presence or absence of polymorphonuclear leukocytes (PMNs) and the bacterial pathogen, Pseudomonas aeruginosa. RESULTS: The CF Airway Chip faithfully recapitulated many features of the human CF airways, including enhanced mucus accumulation, increased cilia density, and a higher ciliary beating frequency compared to chips lined by healthy bronchial epithelial cells. The CF chips also secreted higher levels of IL-8, which was accompanied by enhanced PMN adhesion to the endothelium and transmigration into the airway compartment. In addition, CF Airway Chips provided a more favorable environment for Pseudomonas aeruginosa growth, which resulted in enhanced secretion of inflammatory cytokines and recruitment of PMNs to the airway. CONCLUSIONS: The human CF Airway Chip may provide a valuable preclinical tool for pathophysiology studies as well as for drug testing and personalized medicine.

Electrochemical aptasensor using optimized surface chemistry for the detection of Mycobacterium tuberculosis secreted protein MPT64 in human serum.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. There is a great need for the development of diagnostic tests, which are reliable, sensitive, stable, and low cost to enable early diagnosis of TB in communities with scarce resources. This study reports the optimization and evaluation of a synthetic receptor, an aptamer, for the detection of the secreted protein MPT64, which is a highly immunogenic polypeptide of Mycobacterium tuberculosis, a causative agent of TB. The study investigates combinatorial effects of an aptamer linker and a co-adsorbent onto a gold electrode for optimal binding efficiency and reduced non-specific interactions for label-free detection of MPT64 using electrochemical impedance spectroscopy. Two types of co-adsorbents and two types of aptamer linkers were studied and high specificity and sensitivity to MPT64 was observed for a surface prepared with a thiol PEGylated aptamer HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer and 6-mercaptohexanol in a ratio of 1:100. The developed aptamer-based sensor was successfully used with spiked human serum sample with a limit of detection of 81 pM This work demonstrates the use of the MPT64 aptamer as a lower cost, more sustainable and stable alternative of antibodies for the development of point-of-care TB biosensors decreasing the detection time from several days or hours to thirty minutes.

Reduced graphene-oxide transducers for biosensing applications beyond the Debye-screening limit.

In the field of label-free biosensing, various transducer materials and strategies are under investigation to overcome the Debye-screening limitation of charged biomolecules. We demonstrate an in-line, impedimetric aptasensor with reduced graphene-oxide (rGO) thin films as transducers to detect prostate specific antigens (PSA) in a physiological buffer solution. Unlike classical electrochemical impedance spectroscopy (EIS), this direct, label-free and fully-electronic biosensor approach does not need any redox markers. As specific capture molecules, short anti-PSA aptamers ensured a close binding of the target molecules to the transducer surfaces. Results showed a limit of detection smaller than 33 pM of PSA and a wide detection range from 0.033 to 330 nM fully covering the clinically relevant range of PSA (0.115-0.290 nM). This promising performance can be attributed to the bipolar electronic transport characteristics of the ultra-thin rGO layers similar to pristine graphene. The attachment of target biomolecules to the films changes the resistance of the rGO thin films. Such an in-line EIS configuration with rGO thin films opens promising prospects for biosensing beyond the Debye-screening limitation, which is a major challenge for conventional semiconductor field-effect devices towards clinical applications.

Sensitive and selective Affimer-functionalised interdigitated electrode-based capacitive biosensor for Her4 protein tumour biomarker detection.

A novel Affimer-functionalised interdigitated electrode-based capacitive biosensor platform was developed for detection and estimation of Her4, a protein tumour biomarker, in undiluted serum. An anti-Her4 Affimer with a C-terminal cysteine was used to create the bio-recognition layer via self-assembly on gold interdigitated electrodes for the sensor fabrication. Electrochemical impedance spectroscopy (EIS) in the absence of redox markers was used to evaluate the sensor performance by monitoring the changes in capacitance. The Affimer sensor in buffer and in undiluted serum demonstrated high sensitivity with a broad dynamic range from 1 pM to 100 nM and a limit of detection lower than 1 pM both in buffer and in serum. Furthermore, the Affimer sensor demonstrated excellent specificity with negligible interference from serum proteins, suggesting resilience to non-specific binding. The sensing ability of the present Affimer sensor in spiked undiluted serum suggests its potential for a new range of Affimer-based sensors. The fabricated Affimer sensor can thus be further adapted with other probes having affinities to other biomarkers for a new range of biosensors.

Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serum.

We report the development of a simple and powerful capacitive aptasensor for the detection and estimation of human epidermal growth factor receptor 2 (HER2), a biomarker for breast cancer, in undiluted serum. The study involves the incorporation of interdigitated gold electrodes, which were used to prepare the electrochemical platform. A thiol terminated DNA aptamer with affinity for HER2 was used to prepare the bio-recognition layer via self-assembly on interdigitated gold surfaces. Non-specific binding was prevented by blocking free spaces on surface via starting block phosphate buffer saline-tween20 blocker. The sensor was characterized using cyclic voltammetry, electrochemical impedance spectroscopy (EIS), atomic force microscopy and contact angle studies. Non-Faradic EIS measurements were utilized to investigate the sensor performance via monitoring of the changes in capacitance. The aptasensor exhibited logarithmically detection of HER2 from 1pM to 100nM in both buffer and undiluted serum with limits of detection lower than 1pM. The results pave the way to develop other aptamer-based biosensors for protein biomarkers detection in undiluted serum.

Top-Down Fabricated Silicon Nanowire Arrays for Field-Effect Detection of Prostate-Specific Antigen.

Highly sensitive electrical detection of biomarkers for the early stage screening of cancer is desired for future, ultrafast diagnostic platforms. In the case of prostate cancer (PCa), the prostate-specific antigen (PSA) is of prime interest and its detection in combination with other PCa-relevant biomarkers in a multiplex approach is advised. Toward this goal, we demonstrate the label-free, potentiometric detection of PSA with silicon nanowire ion-sensitive field-effect transistor (Si NW-ISFET) arrays. To realize the field-effect detection, we utilized the DNA aptamer-receptors specific for PSA, which were covalently and site-specifically immobilized on Si NW-ISFETs. The platform was used for quantitative detection of PSA and the change in threshold voltage of the Si NW-ISEFTs was correlated with the concentration of PSA. Concentration-dependent measurements were done in a wide range of 1 pg/mL to 1 µg/mL, which covers the clinical range of interest. To confirm the PSA-DNA aptamer binding on the Si NW surfaces, a sandwich-immunoassay based on chemiluminescence was implemented. The electrical approach using the Si NW-ISFET platform shows a lower limit of detection and a wide dynamic range of the assay. In future, our platform should be utilized to detect multiple biomarkers in one assay to obtain more reliable information about cancer-related diseases.

Hybrid Synthetic Receptors on MOSFET Devices for Detection of Prostate Specific Antigen in Human Plasma.

The study reports the use of extended gate field-effect transistors (FET) for the label-free and sensitive detection of prostate cancer (PCa) biomarkers in human plasma. The approach integrates for the first time hybrid synthetic receptors comprising of highly selective aptamer-lined pockets (apta-MIP) with FETs for sensitive detection of prostate specific antigen (PSA) at clinically relevant concentrations. The hybrid synthetic receptors were constructed by immobilizing an aptamer-PSA complex on gold and subjecting it to 13 cycles of dopamine electropolymerization. The polymerization resulted in the creation of highly selective polymeric cavities that retained the ability to recognize PSA post removal of the protein. The hybrid synthetic receptors were subsequently used in an extended gate FET setup for electrochemical detection of PSA. The sensor was reported to have a limit of detection of 0.1 pg/mL with a linear detection range from 0.1 pg/mL to 1 ng/mL PSA. Detection of 1-10 pg/mL PSA was also achieved in diluted human plasma. The present apta-MIP sensor developed in conjunction with FET devices demonstrates the potential for clinical application of synthetic hybrid receptors for the detection of clinically relevant biomarkers in complex samples.

Highly sensitive dual mode electrochemical platform for microRNA detection.

MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

Label-Free Ultrasensitive Memristive Aptasensor.

We present the very first worldwide ever-reported electrochemical biosensor based on a memristive effect and DNA aptamers. This novel device is developed to propose a completely new approach in cancer diagnostics. In this study, an affinity-based technique is presented for the detection of the prostate specific antigen (PSA) using DNA aptamers. The hysteretic properties of memristive silicon nanowires functionalized with these DNA aptamers provide a label-free and ultrasensitive biodetection technique. The ultrasensitive detection is hereby demonstrated for PSA with a limit of detection down to 23 aM, best ever published value for electrochemical biosensors in PSA detection. The effect of polyelectrolytes on our memristive devices is also reported to further show how positive or negative charges affect the memristive hysteresis. With such an approach, combining memristive nanowires and aptamers, memristive aptamer-based biosensors can be proposed to detect a wide range of cancer markers with unprecedent ultrasensitivities to also address the issue of an early detection of cancer.

DNA aptamer-based sandwich microfluidic assays for dual quantification and multi-glycan profiling of cancer biomarkers.

Two novel sandwich-based immunoassays for prostate cancer (PCa) diagnosis are reported, in which the primary antibody for capture is replaced by a DNA aptamer. The assays, which can be performed in parallel, were developed in a microfluidic device and tested for the detection of free Prostate Specific Antigen (fPSA). A secondary antibody (Aptamer-Antibody Assay) or a lectin (Aptamer-Lectin Assay) is used to quantify, by chemiluminescence, both the amount of fPSA and its glycosylation levels. The use of aptamers enables a more reliable, selective and controlled sensing of the analyte. The dual approach provides sensitive detection of fPSA along with selective fPSA glycoprofiling, which is of significant importance in the diagnosis and prognosis of PCa, as tumor progression is associated with changes in fPSA glycosylation. With these approaches, we can potentially detect 0.5 ng/mL of fPSA and 3 ng/mL of glycosylated fPSA using Sambucus nigra (SNA) lectin, both within the relevant clinical range. The approach can be applied to a wide range of biomarkers, thus providing a good alternative to standard antibody-based immunoassays with significant impact in medical diagnosis and prognosis.

Aptamer-MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen.

This study reports the design and evaluation of a new synthetic receptor sensor based on the amalgamation of biomolecular recognition elements and molecular imprinting to overcome some of the challenges faced by conventional protein imprinting. A thiolated DNA aptamer with established affinity for prostate specific antigen (PSA) was complexed with PSA prior to being immobilised on the surface of a gold electrode. Controlled electropolymerisation of dopamine around the complex served to both entrap the complex, holding the aptamer in, or near to, it's binding conformation, and to localise the PSA binding sites at the sensor surface. Following removal of PSA, it was proposed that the molecularly imprinted polymer (MIP) cavity would act synergistically with the embedded aptamer to form a hybrid receptor (apta-MIP), displaying recognition properties superior to that of aptamer alone. Electrochemical impedance spectroscopy (EIS) was used to evaluate subsequent rebinding of PSA to the apta-MIP surface. The apta-MIP sensor showed high sensitivity with a linear response from 100pg/ml to 100ng/ml of PSA and a limit of detection of 1pg/ml, which was three-fold higher than aptamer alone sensor for PSA. Furthermore, the sensor demonstrated low cross-reactivity with a homologous protein (human Kallikrein 2) and low response to human serum albumin (HSA), suggesting possible resilience to the non-specific binding of serum proteins.