High-resolution spiral microfluidic channel integrated electrochemical device for isolation and detection of extracellular vesicles without lipoprotein contamination.

Recent studies have indicated significant correlation between the concentration of immune checkpoint markers borne by extracellular vesicles (EVs) and the efficacy of immunotherapy. This study introduces a high-resolution spiral microfluidic channel-integrated electrochemical device (HiMEc), which is designed to isolate and detect EVs carrying the immune checkpoint markers programmed death ligand 1 (PD-L1) and programmed death protein 1 (PD-1), devoid of plasma-abundant lipoprotein contamination. Antigen-antibody reactions were applied to immobilize the lipoproteins on bead surfaces within the plasma, establishing a size differential with EVs. A plasma sample was then introduced into the spiral microfluidic channel, which facilitated the acquisition of nanometer-sized EVs and the elimination of micrometer-sized lipoprotein-bead complexes, along with the isolation and quantification of EVs using HiMEc. PD-L1 and PD-1 expression on EVs was evaluated in 30 plasma samples (10 from healthy donors, 20 from lung cancer patients) using HiMEc and compared to the results obtained from standard tissue-based PD-L1 testing, noting that HiMEc could be utilized to select further potential candidates. The obtained results are expected to contribute positively to the clinical assessment of potential immunotherapy beneficiaries.

Electrochemical detection and analysis of tumor-derived extracellular vesicles to evaluate malignancy of pancreatic cystic neoplasm using integrated microfluidic device.

Tumor-derived extracellular vesicles (tdEVs) are one of the most promising biomarkers for liquid biopsy-based cancer diagnostics, owing to the expression of specific membrane proteins of their cellular origin. The investigation of epithelial-to-mesenchymal transition (EMT) in cancer using tdEVs is an alternative way of evaluating the risk of malignancy transformation. An ultra-sensitive selection and detection methodology is an essential step in developing a tdEVs-based cancer diagnostic device. In this study, we developed an indium-tin-oxide (ITO) sensor integrated microfluidic device consisting of two main parts: 1) a multi-orifice flow-fractionation (MOFF) channel for extraction of pure EVs by removing blood cellular debris, and 2) an ITO sensor coupled with a geometrically activated surface interaction (GASI) channel for enrichment and quantification of tdEV. The microfluidic channel and the ITO sensors are assembled with a 3D printed magnetic housing to prevent sample leakage and to easily attach/detach the sensors to/from the microfluidic channel. The tdEVs were successfully captured on the specific antibody modified ITO surfaces in the integrated microfluidic channel. The integrated sensors showed an excellent linear response between 103 and 109 tdEVs/mL. Simultaneous evaluation of the epithelial and mesenchymal markers on the tdEV surfaces successfully revealed the EMT index of the corresponding pancreatic cancer cells. Our ITO sensor integrated microfluidic device showed excellent detection in the clinically relevant tdEVs-concentration range for patients with pancreatic cystic neoplasms. Hence, this system is expected to open a new avenue for liquid biopsy-based cancer prognostics and diagnostics.

Nano-biosensor for the in vitro lactate detection using bi-functionalized conducting polymer/N, S-doped carbon; the effect of αCHC inhibitor on lactate level in cancer cell lines.

A robust amperometric sensor was developed for the lactate detection in the extracellular matrix of cancer cells. The sensor was fabricated by separately immobilizing nicotinamide adenine dinucleotide (NAD+) onto a carboxylic acid group and lactate dehydrogenase (LDH) onto an amine group of bi-functionalized conducting polymer (poly 3-(((2,2':5',2″-terthiophen)-3'-yl)-5-aminobenzoic acid (pTTABA)) composited with N, S-doped porous carbon. Morphological features of the composite layer and sensor performance were investigated using FE-SEM, XPS, and electrochemical methods. The experimental parameters were optimized to get the best results. The calibration plot showed a linear dynamic range between 0.5 µM and 4.0 mM with the detection limit of 112 ± 0.02 nM. The proposed sensor was applied to detect lactate in a non-cancerous (Vero) and two cancer (MCF-7 and HeLa) cell lines. Among these cell lines, MCF-7 was mostly affected by the administration of lactate transport inhibitor, α-cyano-4-hydroxycinnamate (αCHC), followed by HeLa and Vero, respectively. Furthermore, the effect of αCHC concentration and treatment time on the lactate level in the cell lines were demonstrated. Finally, cytotoxicity studies were also performed to evaluate the effect of αCHC on cell viability.

Separation detection of different circulating tumor cells in the blood using an electrochemical microfluidic channel modified with a lipid-bonded conducting polymer.

Different circulating tumor cells (CTCs) in blood were separated and detected through the decoration of anti-cancer drug on the target cells, along with chemical modification of the microfluidic channel walls using a lipid attached covalently to the conducting polymer. The working principle of the electrochemical microfluidic device was evaluated with experimental parameters affecting on the separation, in terms of mass and surface charge of target species, fluid flow rate, AC amplitude, and AC frequency. The separated CTCs were selectively detected via the oxidation of daunomycin adsorbed specifically at the cells using an electrochemical sensor installed at the channel end. The fluorescence microscopic examination also confirmed the separation of CTCs in the channel. To evaluate the reliability of the method, blood samples from 37 cancer patients were tested. The device was able to separate the CTCs with 92.0 ±â€¯0.5 % efficiency and 90.9% detection rate.

Ultrasensitive dual probe immunosensor for the monitoring of nicotine induced-brain derived neurotrophic factor released from cancer cells.

Brain-derived neurotrophic factor (BDNF) was detected in the extracellular matrix of neuronal cells using a dual probe immunosensor (DPI), where one of them was used as a working and another bioconjugate loading probe. The working probe was fabricated by covalently immobilizing capture anti-BDNF (Cap Ab) on the gold nanoparticles (AuNPs)/conducting polymer composite layer. The bioconjugate probe was modified by drop casting a bioconjugate particles composed of conducting polymer self-assembled AuNPs, immobilized with detection anti-BDNF (Det Ab) and toluidine blue O (TBO). Each sensor layer was characterized using the surface analysis and electrochemical methods. Two modified probes were precisely faced each other to form a microfluidic channel structure and the gap between inside modified surfaces was about 19 µm. At optimized conditions, the DPI showed a linear dynamic range from 4.0 to 600.0 pg/ml with a detection limit of 1.5 ±â€¯0.012 pg/ml. Interference effect of IgG, arginine, glutamine, serine, albumin, and fibrinogene were examined and stability of the developed biosensor was also investigated. The reliability of the DPI sensor was evaluated by monitoring the extracellular release of BDNF using exogenic activators (ethanol, K+, and nicotine) in neuronal and non-neuronal cells. In addition, the effect of nicotine onto neuroblastoma cancer cells (SH-SY5Y) was studied in detail.

Detection of Ca2+-induced acetylcholine released from leukemic T-cells using an amperometric microfluidic sensor.

A microfluidic structured-dual electrodes sensor comprising of a pair of screen printed carbon electrodes was fabricated to detect acetylcholine, where one of them was used for an enzyme reaction and another for a detection electrode. The former was coated with gold nanoparticles and the latter with a porous gold layer, followed by electropolymerization of 2, 2:5,2-terthiophene-3-(p-benzoic acid) (pTTBA) on both the electrodes. Then, acetylcholinesterase was covalently attached onto the reaction electrode, and hydrazine and choline oxidase were co-immobilized on the detection electrode. The layers of both modified electrodes were characterized employing voltammetry, field emission scanning electron microscopy, X-ray photoelectron spectroscopy, and quartz crystal microscopy. After the modifications of both electrode surfaces, they were precisely faced each other to form a microfluidic channel structure, where H2O2 produced from the sequential enzymatic reactions was reduced by hydrazine to obtain the analytical signal which was analyzed by the detection electrode. The microfluidic sensor at the optimized experimental conditions exhibited a wide dynamic range from 0.7nM to 1500µM with the detection limit of 0.6 ± 0.1nM based on 3s (S/N = 3). The biomedical application of the proposed sensor was evaluated by detecting acetylcholine in human plasma samples. Moreover, the Ca2+-induced acetylcholine released in leukemic T-cells was also investigated to show the in vitro detection ability of the designed microfluidic sensor. Interference due to the real component matrix were also studied and long term stability of the designed sensor was evaluated. The analytical performance of the designed sensor was also compared with commercially available ACh detection kit.

Enhanced electrochemical sensing of leukemia cells using drug/lipid co-immobilized on the conducting polymer layer.

Electrochemical biosensors using five anticancer drug and lipid molecules attached on the conducting polymer layer to obtain the orientation of drug molecules toward cancer cells, were evaluated as sensing materials and their performances were compared. Conjugation of the drug molecules with a lipid, phosphatidylcholine (PC) has enhanced the sensitivity towards leukemia cells and differentiates cancer cells from normal cells. The composition of each layer of sensor probe was confirmed by electrochemical and surface characterization experiments. Both impedance spectroscopy and voltammetry show the enhanced interaction of leukemia cells using the drug/lipid modified sensor probe. As the number of leukemia cells increased, the charge transfer resistance (Rct) in impedance spectra increased and the amine oxidation peak current of drug molecules in voltammograms decreased at around 0.7-1.0V. Of test drug molecules, raltitrexed (Rtx) showed the best performance for the cancer cells detection. Cancer and normal cell lines from different origins were examined to evaluate the degree of expression of folate receptors (FR) on cells surface, where cervical HeLa cell line was found to be shown the highest expression of the receptor. Impedance and chronoamperometric experiments for leukemia cell line (Jurkat E6-1) showed linear dynamic ranges of 1.0×10(3)-2.5×10(5) cells/mL and 1.0×10(3)-8.0×10(3) cells/mL with detection limits of 68±5 cells/mL and 21±3 cells/mL, respectively.

Sensitive NADH detection in a tumorigenic cell line using a nano-biosensor based on the organic complex formation.

A robust amperometric sensor for ß-nicotinamide adenine dinucleotide (NADH) detection was developed through the organic complex formation with ethylenediaminetetraacetic acid (EDTA) bonded on the polyethylenimine (PEI)/activated graphene oxide (AGO) layer. The EDTA immobilized sensor probe (GCE/AGO/PEI-EDTA) revealed a catalytic property towards NADH oxidation that allows for the highly sensitive electrochemical detection of NADH at a low oxidation potential. Surface characterization demonstrated that the negatively charged AGO acted as nanofillers in the positively charged PEI matrix through the charge interaction. The immobilization of EDTA on the polymer layer provided more surface area for NADH to interact with through the enhanced chemical interlocking between them. We observed the strong interaction between NADH and EDTA on the AGO/PEI layer using a quartz crystal microbalance (QCM), X-ray photoelectron spectroscopy (XPS), and the calculation of the minimized energy for complex formation. The dynamic range of NADH was determined to be between 0.05µM and 500µM with a detection limit (LD) of 20.0±1.1nM. The reliability of the developed sensor for biomedical applications was examined by detecting NADH in tumorigenic lung epithelial cells using the standard addition method.

Amperometric sensing of HIF1α expressed in cancer cells and the effect of hypoxic mimicking agents.

Hypoxia inducible factor 1 alpha (HIF1α) overexpression was detected in cancerous cells using an amperometric immunosensor with a nano-bioconjugate. The sensor probe was fabricated by covalently immobilizing the antibody (anti-HIF1α) onto a composite layer of functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA) formed on a layer of gold nanoparticles (AuNPs). A nano-bioconjugate with hydrazine and a secondary antibody of HIF1α (sec-Ab2) attached on AuNPs reveals the immunoreaction at the sensor probe through the catalytic reduction of H2O2 by hydrazine at -0.35V vs. Ag/AgCl. Morphology and performance of the sensor probe were characterized using FE-SEM, XPS, EIS, and cyclic voltammetry. The calibration plot at optimized experimental conditions shows a dynamic range of 25-350pM/mL with a detection limit of 5.35±0.02pM/mL. The reliability of the sensor was evaluated using non-cancerous Vero and cancerous MCF-7 cell lysates, where the HIF1α expression was compared with three cancerous cell lines MCF-7, PC-3, and A549. Furthermore, the sensor probe confirms the stable expression of HIF1α in the A549 lung cancer cells when exposing them to hypoxic mimicking agents Co, Ni, and Mn ions. Of these, Co ions show the highest stabilization effect on HIF1α followed by Ni and Mn ions, respectively.

Ultrasensitive cytosensing based on an aptamer modified nanobiosensor with a bioconjugate: Detection of human non-small-cell lung cancer cells.

A novel aptamer-based amperometric nanobiosensor was designed for the sensitive and selective detection of A549 human non-small-cell lung cancer (NSCLC) cells. The cytosensing was performed using a MUC1 aptamer probe with a bioconjugate, where the probe was fabricated by the covalent immobilization on a conducting polymer nanocomposite formed through the self-assembly of 4-([2,2':5',2''-terthiophen]-3'-yl) benzoic acid (TTBA) on AuNPs. A bioconjugate composed of hydrazine and aptamer attached on AuNPs was used to reveal the selectively amplified detection signal. The cells were quantitatively analyzed using chronoamperometric measurements, and the results were further compared and confirmed using microscopic and DPV methods based on silver staining cytosensing experiments. The proposed aptasensor showed a high affinity for MUC1 positive lung cancer cells (A549) compared with the other control cancer cells, including human prostate (PC3), MUC1 negative normal lung (MRC-5), and liver tumors (HepG2) cells. An excellent dynamic range of the proposed method was obtained from 15 to 1×10(6) cells/mL with a detection limit of 8 cells/mL.

An amperometric nanobiosensor for the selective detection of K⁺-induced dopamine released from living cells.

A highly sensitive amperometric sensor has been studied for selective monitoring of K(+)-induced dopamine released from dopaminergic cells (PC12) which is based on an EDTA immobilized-poly(1,5-diaminonaphthalne) (poly-DAN) layer comprising graphene oxide (GO) and gold nanoparticles (GO/AuNPs). The integration of a negatively charged probe molecule on the poly-DAN/GO/AuNPs nanohybrid attained the signal enhancement to discriminate dopamine (DA) molecules from foreign species by catalytic effect and surface charge, and hydrogen bonding-based interactions with a probe molecule. The sensor performance and morphology were investigated using voltammetry, impedance spectrometry, SEM, and XPS. Experimental variables affecting the analytical performance of the sensor probe were optimized, and linear response was observed in the range of 10 nM-1 µM with a detection limit of 5.0 nM (±0.01) for DA. Then, the sensor was applied to monitor dopamine released from PC12 cells upon extracellular stimulation of K(+) ions. It was also confirmed that K(+)-induced dopamine release was inhibited by a calcium channel inhibitor (Nifidipine). The results demonstrated that the presented biosensor could be used as an excellent tool for monitoring the effect of exogenous agents on living cells and drug efficacy tests.