Label-free Electrochemical Impedance Research Articles

Electrografted Laser-Induced Graphene: Direct Detection of Neurodegenerative Disease Biomarker in Cerebrospinal Fluid.

There are more than 50 neurodegenerative disorders, and amyotrophic lateral sclerosis (ALS) is one of the most common disorders that poses diagnostic and treatment challenges. The poly glycine-proline (polyGP) dipeptide repeat is a toxic protein that has been recognized as a pharmacodynamic biomarker of C9orf72-associated (c9+) ALS, a subtype of ALS that originates from genetic mutation. Early detection of polyGP will help healthcare providers start timely gene therapy. Herein, we developed a label-free electrochemical immunoassay for the simple detection of polyGP in unprocessed cerebrospinal fluid (CSF) samples collected from ALS patients in the National ALS Biorepository. For the first time, an electrografted laser-induced graphene (E-LIG) electrode system was employed in a sandwich format to detect polyGP using a label-free electrochemical impedance technique. The results show that the E-LIG-modified surface exhibited high sensitivity and selectivity in buffer and CSF media with limit of detection values of 0.19 and 0.27 ng/mL, respectively. The precision of the calibration model was better in CSF than in the buffer. The E-LIG immunosensor can easily select polyGP targets in the presence of other dipeptide proteins translated from the c9 gene. Further study with CSF samples from ALS patients demonstrated that the label-free E-LIG-based immunosensor not only quantified polyGP in the complex CSF matrix but also distinguished between c9+ and non-c9- ALS patients.

Deaeration of incubation buffer to minimize electrochemical impedance spectroscopy signal drift on screen-printed gold electrodes and its use for label-free detection of vancomycin

Disposable screen-printed gold electrodes (SPAuEs) are used for the development of numerous immunosensors owing to their ease of modification with biorecognition molecules. A major limitation in the development of SPAuEs based biosensors is the single-use of the electrodes leading to higher costs for sensor development, whereas multiple measurements can affect the sensor stability and reproducibility. Especially, label-free electrochemical impedance spectroscopy (EIS) based SPAuEs sensors are prone to signal drift resulting in false-positive or false-negative data questioning the reliability of the assay. We found that repeated EIS measurements on SPAuEs in ferri- and ferrocyanide solution yielded a significant increase in resistance to charge transfer (Rct) value (positive signal drift) even in a control solution (without the target analyte). The positive signal drift follows the typical pattern of a concentration-dependent calibration curve, rendering repeated measurement error-prone for sensing purposes. In this study, it is shown that the incubation of the SPAuEs in nitrogen purged deaerated phosphate buffered saline (N-PBS) minimized this signal drift in repeated/multiple EIS measurements. This method was then used to develop a peptide-based EIS biosensor for the detection of Vancomycin, a last-line antibiotic used for the treatment of severe multidrug-resistant bacterial infection. This protocol can be followed for the biosensing of other clinically important biomarkers.

Development of an Integrated Multi-Method Electrochemical Biosensor for Rapid-on-Site Detection and Quantification of Cyanotoxins

The accumulation of Cyanotoxins (CTXs) in water reservoirs is an emerging global concern. Exposure to microcystin-LR (MC-LR), among the most common CTXs, is associated with hepatotoxicity and carcinogenesis and leads to both acute and chronic damages. Stringent regulation has recently been imposed by the World Health Organization, limiting the allowed MC-LR concentration in drinking water to <1 μg/L. Current methods for MC-LR detection require expensive equipment, trained personnel, and labor-intensive preparation. Therefore, there is an urgent need for an affordable, on-site diagnostic tool providing a rapid quantitative determination of MC-LR in various water types. In this study, we have developed an electrochemical (EC) biosensor that comprised of a miniaturized and multiplexed microchips, functionalized with anti-MC-LR antibodies, and MC-LR detection is based on the transduction of bimolecular binding into an electrochemical signal. The transduction method applied is based on the comparison of two (2) electrochemical techniques: a label-free Electrochemical Impedance Spectroscopy (EIS) a labeled Electrochemical Immunoassay (ECI) detection of MC-LR in <10 min. The former, the EIS-based detection method, demonstrated the feasibility of detecting ultra-low (3 ppt or ng/L) MC-LR concentrations, and preliminary results also showed that the biochip demonstrated its use in rapid quantitative detection of microcystins in raw cyanobacterial cultures. However, the latter, ECI-based detection method showed a detection limit of 3µg/L (3ppb) of purified MC-LR concentrations, which is an order of magnitude less sensitive than the EIS-based detection method. These LODs obtained from the two methods are superior to currently employed solid-phase immunoassays and other analytical methods. Based on our findings, we anticipate that in the near future electrochemical biosensors would be an essential tool in water monitoring and environmental diagnostics. Figure 1

Label-Free Detection of CA19-9 Using a BSA/Graphene-Based Antifouling Electrochemical Immunosensor.

Evaluating the levels of the biomarker carbohydrate antigen 19-9 (CA19-9) is crucial in early cancer diagnosis and prognosis assessment. In this study, an antifouling electrochemical immunosensor was developed for the label-free detection of CA19-9, in which bovine serum albumin (BSA) and graphene were cross-linked with the aid of glutaraldehyde to form a 3D conductive porous network on the surface of an electrode. The electrochemical immunosensor was characterized through the use of transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscope (AFM), UV spectroscopy, and electrochemical methods. The level of CA19-9 was determined through the use of label-free electrochemical impedance spectroscopy (EIS) measurements. The electron transfer at the interface of the electrode was well preserved in human serum samples, demonstrating that this electrochemical immunosensor has excellent antifouling performance. CA19-9 could be detected in a wide range from 13.5 U/mL to 1000 U/mL, with a detection limit of 13.5 U/mL in human serum samples. This immunosensor also exhibited good selectivity and stability. The detection results of this immunosensor were further validated and compared using an enzyme-linked immunosorbent assay (ELISA). All the results confirmed that this immunosensor has a good sensing performance in terms of CA19-9, suggesting its promising application prospects in clinical applications.

A diagnostic electrochemical aptasensor development for sCD80 protein detection in human serum

Elevating soluble CD80 (sCD80) in human serum is a natural response to autoimmune diseases such as rheumatoid arthritis (RA). The level of sCD80 is associated with RA development and prognosis; therefore, it is potentially used as a biomarker. sCD80 is commonly measured in human serum using immunoassays (e.g., ELISA) with multiple drawbacks, mainly cross-reactivity. Aptamer-based biosensors (aptasensors) development for quantifying and detecting different biological molecules demonstrates applicability in next-generation medicine and biomarker detection. Herein, we selected a specific aptamer for sCD80 by conventional in-vitro selection process (SELEX) with the high-affinity aptamer (Kd = 47.69 nM). A sensitive aptasensor, for the first time, was developed on a screen-printed gold electrode (AuSPE) platform compatible with easy-to-use label-free electrochemical impedance spectroscopy. The immobilization of the aptamer on the gold surface and the presence of sCD80 in a complex with the aptamer were characterized by photo-induced force microscopy, which revealed the uniform assembly of the aptamer monolayer and the distribution of sCD80 on the electrode surface. The developed aptasensor showed a linear performance (0.025–10.0 nM of protein) with a detection limit of 8.0 pM. Furthermore, the aptasensor was tested in a biological matrix, where a linear signal was observed for the increased amount of spiked sCD80 (R2 = 0.9887). The recovery of the spiked amounts ranged from 105 to 125% with coefficient of variation (CV%) <7%, which supported the applicability of this sensor in detecting sCD80 for diagnosis.

Label-free electrochemical biosensor based on Ng-PCL polymer signal amplification for the detection of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is the related antigen first extracted from embryonic tissue and is an acidic glycoprotein with human embryonic antigenic properties. It is an important biomarker for the detection of many malignancies. In this paper, we have developed a signal amplified sandwich-type electrochemical impedance biosensor based on naringin initiated poly(ε-caprolactone) (Ng-PCL) polymer for highly sensitive and rapid determination of CEA. The polymer Ng-PCL is a macromolecular polymer that can increase the sensitivity of biosensor. First, SH-functionalized CEA-aptamer 1 (Apt1) was attached to the electrode surface as a recognition probe by Au-S bonds in order to form a self-assembly monolayer (SAM). Then, the one end of CEA antigen could recognize the Apt1 modified on the electrode surface, the other end could recognize the COOH-functionalized CEA-aptamer 2 (Apt2) forming a sandwich structure. Finally, polymer Ng-PCL as a signal amplification unit, which was connected to Apt2 by ester bonds. As a result, the electron transfer in the electrode interface was hindered effectively and a label-free electrochemical impedance sensor was constructed successfully. The high sensitivity and selectivity of biosensor relied on the SAM, polymer Ng-PCL, and specific recognition of CEA. Under the optimum experimental conditions, we proposed electrochemical impedance biosensor had wide detection range between 5 pg/mL and 200 ng/mL, and the sensitivity can reach 0.675 pg/mL. Moreover, the electrochemical impedance biosensor performed high specificity, good stability and satisfactory reproducibility as well. Thus, this strategy will provide new path for the detection of other biomarkers in early clinical diagnosis.

Novel Label-Free Electrochemical Impedance Immunosensor Using a Three-Dimensional Flower-Shaped Gold Nanoparticle@Molybdenum(IV) Sulfide/Graphene Composite for the Ultrasensitive Determination of Procalcitonin

Procalcitonin (PCT) is an inflammatory marker with significance in the early diagnosis of sepsis. The label-free electrochemical impedance immunosensor has good prospects for the determination of this disease marker due to its cost-effectiveness, high specificity and simple operation. However, developing an immunosensor for procalcitonin faces challenges due to the absence of satisfactory electrodes. Herein, a novel label-free electrochemical impedance immunosensor using the three-dimensional (3D) flower-shaped gold nanoparticle@molybdenum(IV) sulfide/graphene (Au NPs@MoS2/graphene) composite is reported for procalcitonin. Under the optimal conditions, the immunosensor presents a linear response from 10 fg mL−1 to 50 ng mL−1 and an ultra-low detection limit of 2.3 fg mL−1 for for procalcitonin. The excellent performance is due to the unique 3D flower-shape and the synergistic effect of Au NPs@MoS2/graphene, generating a large surface area, high electrical conductivity, and increased immobilization of anti-PCT. Additionally, the immunosensor exhibits suitable specificity, stability, and reproducibility, and was used to determine procalcitonin in serum which may allow clinical screening and diagnosis of sepsis.

Sensitive detection of PARP-1 activity by electrochemical impedance spectroscopy based on biomineralization

Poly(ADP)ribose polymerase-1 (PARP-1) has attracted much attention as a tumor marker in recent years. Based on the large negative charge and hyperbranched structure of PARP-1 amplified products (PAR), many detection methods have been established. Herein, we proposed a label-free electrochemical impedance detection method based on the large amount of phosphate groups (PO43−) on the surface of PAR. Although EIS method has high sensitivity, it is not sensitive enough to discern PAR effectively. Therefore, biomineralization was incorporated to increase the resistance value (Rct) distinctly because of the poor electrical conductivity of CaP. During biomineralization process, plentiful Ca2+ was captured by PO43− of PAR through electrostatic interaction, resulting in an increasing Rct of modified ITO electrode. In contrast, when PRAP-1 was absent, only a little Ca2+ was adsorbed on the phosphate backbone of the activating dsDNA. As a result, the biomineralization effect was slight and only a negligible Rct change occurred. Experiment results showed that Rct was associated closely with the activity of PARP-1. There was a linear correlation between them when the activity value was in the range of 0.005–1.0 U. The calculated detection limit was 0.003 U. Results of real samples detection and the recovery experiments were satisfactory, indicating the method has an excellent application prospect.

Label-free biosensor for trace insulin-like growth factor-I assay based on rGO-SnS2 heterostructure nanocomposite

Insulin-like growth factor-I (AgIGF-I) is a key biomarker in tumor progression. In this work, a novel label-free electrochemical impedance biosensing platform for detection of AgIGF-I was developed on the basis of a nanocomposite of reduced graphene oxidetin sulfide (rGO-SnS2) loaded with gold nanoparticles (AuNPs). The rGO-SnS2 surface serves as an interfacial matrix, exhibiting outstanding electrochemical activity because of its large surface area; meanwhile, the abundant active sites in rGO-SnS2 promote AuNP deposition and improve antibody (Ab) immobilization. The specific recognition between IGF-I antibody (AbIGF-I) and antigen (AgIGF-I) at the interface induces changes in electrochemical impedance, which can distinguish AgIGF-I from a complex biological media to ensure the selectivity. Electron transfer resistance (Rct) corresponds to the number of analytes on the interface, which serves as the basis for the design of an electrochemical impedance biosensor. Under optimized conditions, the designed biosensor exhibits a linear response range of 0.25–750.0 pg/mL and an ultralow detection limit of 0.12 pg/mL (S/N = 3), and the results based on an electrochemical method are consistent with those of enzyme-linked immunosorbent assay (ELISA). Such an electrochemical biosensor is highly effective for IGF-I sensing in human plasma samples, which broadens the application of the heterostructure [email protected]2 nanocomposite in bioanalysis and clinical diagnosis.

Development of label-free electrochemical impedance spectroscopy for the detection of HLA-B*15:02 and HLA-B*15:21 for the prevention of carbamazepine-induced Stevens-Johnson syndrome

BackgroundCarbamazepine (CBZ) is an FDA-approved anticonvulsant that is widely used to treat epilepsy, bipolar disorder, trigeminal neuralgia and chronic pain. Several studies have reported a strong association between HLA-B*15:02 and carbamazepine-induced Stevens–Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN). However, the HLA-B75 serotype (HLA-B*15:02, HLA-B*15:08, HLA-B*15:11 and HLA-B*15:21) has been found in patients with carbamazepine-induced SJS/TEN. MethodsThis study aimed to develop label-free electrochemical impedance spectroscopy (EIS) for the detection of HLA-B*15:02 and HLA-B*15:21 after PCR-SSP amplification. A total of 208 DNA samples were tested. The impedance was measured and compared to standard gel electrophoresis. ResultsThe developed label-free EIS identified HLA-B*15:02 and HLA-B*15:21 alleles with 100% sensitivity (95% CI: 86.773%–100.000%) and 95.05% specificity (95% CI: 90.821%–97.714%), comparable to commercial DMSc 15:02 detection kits. ConclusionsWe successfully developed a novel PCR-SSP associated with signal impedance changes to detect the HLA-B*15:02 allele and HLA-B*15:21 without downstream amplicon size analysis that is suitable for screening individuals before indication of CBZ therapy.

2D Carbon Nitride-Based Electrochemical Aptasensor for Label-Free and Highly-Sensitive Detection of Okadaic Acid in Shellfish

Okadaic acid (OA) is a marine toxin accumulated in bivalves causing severe diarrhetic shellfish poisoning, which has become a huge threaten to human health, food safety, and environmental protection. Therefore, it is highly essential to develop cost-effective and convenient approaches for OA detection. Recent advances in the electrochemical transducer and nanomaterials may provide novel approaches to address this issue. Herein, a label-free electrochemical impedance aptasensor was developed for the sensitive detection of OA in shellfish. Two-dimensional nanomaterials (carbon nitride) conjugated with aptamers were employed as the sensitive element for OA detection, which have been well characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Fourier Transform Infrared Spectrometer (FTIR) and X-ray diffraction (XRD). The obtained aptasensor exhibited a good performance for the OA detection with a wide linearity ranging from 1 × 10−14 mol l−1 to 1 × 10−8 mol l−1. The limit of detection was down to 1 × 10−14 mol l−1. Besides, this aptasensor had a good selectivity towards OA in the presence of other toxins, such as dinophysistoxins (DTX), pectenotoxins (PTX), and yessotoxin (YTX). Meanwhile, it also showed a good reproducibility and stability. The real mussel samples results showed a good recovery rate. The simple and cost-effective sensing strategy to marine toxins could be applied in the fields of seafood safety and water quality control.

A Label-Free Electrochemical Impedimetric Immunosensor with Biotinylated-Antibody for SARS-CoV-2 Nucleoprotein Detection in Saliva.

The timely detecting of SARS-CoV-2 coronavirus antigens for infection validation is an urgent request for COVID-19 pandemic control. This study constructed label-free electrochemical impedance spectroscopy (EIS)-based immunosensors based on gold nanostructured screen-printed carbon electrodes (AuNS/SPCEs) to detect the SARS-CoV-2 nucleocapsid protein (N-protein) in saliva. Using short-chain 3-mercaptopropionic acid (MPA) as a linker to covalently bond streptavidin (SA) and bovine serum albumin (BSA) for controlling the oriented immobilization of the biotinylated anti-N-protein antibody (BioAb) can offer a greater sensitivity, a lower limit of detection (LOD), and better reproducibility of immunosensors (defined as BioAb/SA-BSA/MPA/AuNS/SPCEs) than the antibody randomly immobilized immunosensors and the long-chain 11-mercaptoundecanoic acid (MUA)-modified immunosensors (BioAb/SA-BSA/MUA/AuNS/SPCEs). The BioAb/SA-BSA/MPA/AuNS/SPCE-based immunosensors presented good linearity from 0.01 ng/mL to 100 ng/mL and a low LOD of 6 pg/mL in a phosphate buffer solution (PBS) and PBS-diluted saliva. Moreover, the immunosensor exhibited little cross-activity with other viral antigens such as MERS-CoV N-protein, influenza A N-protein, influenza B N-protein, and SARS-CoV-2 spike protein, indicating the high specificity of the immunosensors. The disposable label-free EIS-based immunosensors have promising potential in facilitating the rapid and sensitive tests of saliva-based COVID-19 diagnostics.

A Label-Free Electrochemical Impedance Genosensor Coupled with Recombinase Polymerase Amplification for Genetically Modified Maize Detection

As the cultivation scale of genetically modified (GM) crops strongly increases, a convenient DNA assay is highly demanded in resource-limited areas. A label-free electrochemical impedance (EI) genosensor using gold carbon dots (GCDs) was developed with easy-to-use portable device. GCDs were used to modify screen-printed carbon electrode and immobilize capture probes by conducting a simple protocol. After the amplification products anchored on the sensor surface via hybridization reactions, the EI signal increased due to the formation of biocomplex hampering the interfacial electron transfer. Under the optimal conditions, the proposed genosensor coupled with recombinase polymerase amplification (RPA) could detect maize Ruifeng12-5 in a linear range of 0.10–5.0% with a detection limit of 0.10%. In addition, combined with a one-step extraction and RPA amplification, the proposed sensor device can be applied in resource-limited laboratories without expensive instruments or professionals. Therefore, the developed method provides an easy-to-use and sensitive platform for GM organism detection.

Fetuin derivatised surface for evaluation of neuraminidase inhibitors of Peste des petits ruminants virus on electrochemical impedance sensor

Neuraminidase (NA) is a viral glycoprotein present in the surface of viruses belonging to paramyxoviridae and orthomyxoviridae. It helps in viral attachment and movement in the airways. It is also essential for release of viral progeny from the infected cells. Thus, anti-neuraminic agents have the potential to obstruct infection well before the virus interacts with the host. In the present study, we describe a label-free electrochemical impedance spectroscopy to characterise the NA inhibitors of plant origin against the Peste des petits ruminants virus NA. A decrease in charge transfer resistance, ΔRct (>50%;p<0.05) was observed for morin and eugenol when incubated against NA validates their anti-neuraminidase activity. Further, reduced virus propagation in vitro corroborated the efficient anti-neuraminic activity of morin and eugenol. PPRV Neuraminidase Inhibition stipulated the IC50 of 26.01 µM and 115.1 µM for morin and eugenol respectively. Additionally, we observed a selective index of both phytochemicals (16.2 for morin and 11.75 for eugenol). The proposed impedance spectroscopy method has sensitivity comparable to that of traditional, but avoiding the tedious screening system for anti- neuraminic compounds.

Determination of Vascular Endothelial Growth Factor (VEGF) in Cell Culture Medium by Gold-Coated Magnetic Nanoparticle Based Label-Free Electrochemical Impedance Spectroscopy (EIS)

A gold-coated magnetic nanoparticle (MNPs@Au) based label-free electrochemical impedance spectroscopy (EIS) sensor is reported for determining vascular endothelial growth factor (VEGF) in cell culture media. VEGF antibodies (Ab) were modified on MNPs@Au to construct an Ab-bound magnetic complex (MNPs@Au-Ab) that was magnetically immobilized on a magnetic glassy carbon electrode (mGCE). The VEGF is captured on the MNPs@Au-Ab/mGCE, leading to increased electron-transfer resistance (R et) utilized for the determination of VEGF. Combining the highly efficient immobilization of antibodies on magnetic nanocomposites with the high sensitivity of electrochemical impedance spectroscopy, an excellent dynamic range for the VEGF concentration from 1.0 × 10−2 to 1.0 × 104 ng mL−1 was obtained with a detection limit of 2.43 pg mL−1. Quantitative determination and the investigation of the secretion of VEGF by various cells were also conducted. VEGF released by cancer cells (MDA-MB-231 cells and HCT116 cells) was significantly higher than for normal cells (L02 cells), which matched well with an enzyme linked immunosorbent assay (ELISA). These results show that the secretion of VEGF is associated with the metastatic level of cells. The reported sensor is expected to be applied in studies of cancer and tumor metastasis.

A new class of sensing elements for sensors: Clamp peptides for Zika virus

The design of a new class of selective and high affinity antibody mimetics termed clamp peptide (CP) that incorporate three short peptides structurally and mechanically mimicking a clamp is proposed as sensing elements for a reliable detection sensor platform. The CPs consist of two short peptides functioning as arms that recognize two different epitopes in the target protein and are connected by a third short peptide that acts as a hinge between the peptide arms. For the construction of CPs, we employed a rational design combined with computational methods. To illustrate our approach, we designed a CP that binds selectively to the envelope protein of the Zika virus (ZIKV). The virtual docking cycles were run maximizing the discrimination between ZIKV and Dengue virus (DENV) envelope proteins. DENV was chosen among the flavivirus family because it has high structural similarity with ZIKV. When employed in a colorimetric binding assay or in label-free electrochemical impedance sensor format, the CP was selective for ZIKV vs DENV particles showing detection limit under 104 copies/mL, comparable to anti-ZIKV antibodies.Apparent dissociation binding constants (Kd) confirmed a better performance of CPs than mono-arm peptides (Kd of best CP = 162 nM ± 23 nM; Kd of best mono-arm peptide = 11.15 ± 2.76 μM).The performance of the assays based on CPs was also verified in serum and urine (diluted 1:10 and 1:1 respectively). The detection limits of CPs decreased about one order of magnitude for ZIKV detection in serum or urine, with a distinct analytical signal starting from 105 copies/mL of ZIKV.

Impedimetric Detection of Femtomolar Levels of Interleukin6, Interleukin 8, and Tumor Necrosis Factor Alpha Based on Thermally Modified Nanotubular Titanium Dioxide Arrays.

An inexpensive, easy to prepare, and label-free electrochemical impedance spectroscopy-based biosensor has been developed for the selective detection of human interleukin 6 (IL-6), interleukin 8 (CXCL8, IL-8), and tumor necrosis factor (TNFα)—potential inflammatory cancer biomarkers. We describe a, so far, newly developed and unexplored method to immobilize antibodies onto a titanium dioxide nanotube (TNT) array by physical adsorption. Immobilization of anti-IL-6, anti-IL-8, and anti-TNFα on TNT and the detection of human IL-6, IL-8, and TNFα were examined using electrochemical impedance spectroscopy (EIS). The impedimetric immunosensor demonstrates good selectivity and high sensitivity against human biomarker analytes and can detect IL-6, IL-8, and TNFα at concentrations as low as 5 pg/mL, equivalent to the standard concentration of these proteins in human blood. The calibration curves evidenced that elaborated biosensors are sensitive to three cytokines within 5 ÷ 2500 pg/mL in the 0.01 M phosphate-buffered saline solution (pH 7.4).

An analysis method to detect the presence of DNA using electrochemical impedance spectroscopy (EIS) for real-time PCR

This paper reports a technique for detecting the presence of DNA in real-time PCR using label-free electrochemical impedance spectroscopy. The change in the real and imaginary parts of the impedance with the number of PCR cycles indicates the concentration of amplified DNA. In general, the real part of the impedance tends to increase with the number of PCR cycles over most of the measurement frequency range (100 Hz–1 MHz), whereas the imaginary part of the impedance increases in the negative direction. The optimal frequency for impedance analysis is determined by the performance index (PI), as one of the figures of merit, which quantitatively indicates how monotonously the impedance changes with the PCR cycle number. The detectability of DNA in the sample is scrutinized by analyzing the impedance at the optimal frequency (3.984 kHz and 20.02 kHz for the real and imaginary parts of the impedance, respectively) obtained from the performance index. The proposed technique can be used to quantitatively analyze PCR by applying simple electrochemical impedance spectroscopy (EIS) technology so that a label-free real-time monitoring system has high potential for a portable PCR system.

Ultrasensitive label-free detection for lung cancer CYFRA 21-1 DNA based on ring-opening polymerization

Cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA is perceived as sensitive tumor marker for the diagnosis of non-small cell lung cancer and other tumor. Herein, linear chain poly(ε-caprolactone) (PCL) synthesized by ring-opening polymerization is applied to ultrasensitive label-free electrochemical impedance detection system for CYFRA 21-1 DNA. First, thiolated peptide nucleic acid (PNA) is self-assembled into the Au electrode surface through the formation of Au–S bonds, allowing the PNA to act as biomolecular probe and form PNA/DNA heteroduplex with the target DNA via specific hybridization. Then, PCL is conjugated to the immobilized DNA on the electrode via “carboxylate–Zr4+–phosphate” bridges. Finally, the electrochemical response of modified PNA/DNA/Zr4+/PCL electrode is determined by electrochemical impedance method to quantify of CYFRA 21-1 DNA. Under optimal conditions, this method exhibits highly sensitivity with a broad linear range (0.1 fM – 1 nM) (R2 = 0.995) and the limit of detection (LOD) is as low as 10.73 aM, which is equivalent to just 64 molecules in a 10 μL sample. What's more, the high selectivity, good anti-interference, label-free operation, and real-time monitoring in complex samples of the proposed strategy demonstrate its broad application for the early diagnosis and clinical monitoring.

Hyaluronic acid-based label-free electrochemical impedance analysis for cancer cell quantification and CD44 expression

In this study, a novel label-free electrochemical impedance spectroscopy (EIS) sensor was developed by successfully conjugating hyaluronic acid (HA) with bovine serum albumin (BSA)-modified gold nanoparticles (GNPs) for cancer cell quantification and CD44 expression analysis. Based on the molecular recognition of HA, the CD44-overexpressing cancer cells can be captured on the nanocomposites (HA-BSA-GNPs) modified electrode, increasing the electron-transfer resistance (Ret). The change of Ret was measured by the EIS to quantify the number of cancer cells and evaluate the CD44 expression level on the cell surface. Under optimal conditions, the change of Ret was proportional to the logarithm of the cell concentration, namely from 2.0 × 102 to 3.0 × 105 cells mL−1 with a detection limit of 128 cells mL−1 for the MDA-MB-231 cells. Moreover, compared to HCT116 and L02 cells, the MDA-MB-231 cells exhibited a distinctly higher expression level of CD44. This indicates that the MDA-MB-231 cells have greater metastatic potential. The proposed sensor may find potential applications in cancer monitoring and research on tumor metastasis.