Highly sensitive detection of cancer cells based on the DNA barcode assay and microcapillary electrophoretic analysis

Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (μCE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the μCE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF‐7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the μCE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF‐7 and SW620 cells in a volume of 200 μL, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the μCE analysis.     

Single Microbead‐Anchored Fluorescent Immunoassay (SMFIA): A Facile and Versatile Platform Allowing Simultaneous Detection of Multiple Antigens

A new concept of single microbead (MB)‐anchored fluorescent immunoassay (SMFIA) is proposed with greatly improved sensitivity. In the SMFIA, a single MB is manipulated as the reaction carrier so that the target‐tethered fluorescent immunocomplexes will be highly concentrated on one MB. By monitoring the enriched fluorescence signal on the single MB through imaging, highly sensitive target quantification can be realized just by employing the most common sandwich immunoreactions without requirement of further signal amplification routes. The high sensitivity of the SMFIA can fully meet the demand of current medical diagnosis. Furthermore, we have further advanced a fluorescence‐encoding mechanism for the proposed SMFIA which allows the simultaneous detection of multiple antigens in a single reaction. Sharing the distinct advantages of simple operation, high sensitivity and multiplexed detection capability, the SMFIA provides a general platform for the detection of various biomarkers.     

Carrier-resolved technology for homogeneous and multiplexed DNA assays in a 'one-pot reaction'

For clinical diagnosis, a small number of targets (2-10 biomarkers) are often all that is required for disease assessment and accurate early disease diagnosis. In the current paper we have developed novel, carrier-resolved, single-label-based multiplexed assays for the simultaneous detection and quantification of a limited number of DNA targets associated with breast cancer. In contrast to current encoding strategies, every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physico-chemical signature. Moreover, a simple chemiluminescence setup is employed to read the carrier code instead of expensive and complicated flow-cytometer or imaging-systems commonly used for multiplexed assays. Herein we demonstrate a new protocol using three homogeneous carriers, i.e. thermo-sensitive poly(N-isopropylacrylamide) (PNIP), polystyrene beads, and magnetic beads respectively. This new methodology allowed for the simultaneous determination of three oligonucleotide sequences (60 bases in length) associated with the breast cancer gene (BRCA1) and showed high selectivity and attomolar-femtomolar sensitivity. The mixture of three different capture probe conjugates first hybridizes with three corresponding target sequences, sandwiches with three biotinylated DNAs, and then reacts with peroxidase-streptavidin polymer in a single vessel without any washing, leading to the development of a 'one-pot reaction system'. Only one washing step in our protocol is required prior to detection leading to our whole procedure being simple and efficient. The results show that the hybridization response to sample mixtures containing increasing levels of each target is proportional to the amount of corresponding DNA targets, indicating minimal cross-interferences. The work presented here validates the design and concept of a system for the detection of a limited number of DNA targets and provides the foundation for the development of highly sensitive techniques with increased multi-analyte capabilities.     

Multiplexed Determination of Amino‐Terminal Pro‐B‐Type Natriuretic Peptide and C‐Reactive Protein Cardiac Biomarkers in Human Serum at a Disposable Electrochemical Magnetoimmunosensor

A rapid magnetoimmunosensor for the simultaneous determination of two cardiac biomarkers, amino‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) and C‐reactive protein (CRP), in human serum is described. Specific capture antibodies were covalently immobilized onto carboxylic acid‐modified magnetic beads. The quantification of NT‐proBNP and CRP was performed by using indirect competitive and sandwich configurations, respectively, and horseradish peroxidase‐labeled tracers. The use of dual screen‐printed carbon electrodes allowed the achievement of simultaneous independent amperometric readout for each cardiac biomarker. The developed methodology showed very low limits of detection (0.47 ng mL^?1). An international standard for CRP serum spiked with NT‐proBNP was analyzed to evaluate the usefulness of the magnetoimmunosensor.     

Ultrasensitive electrochemical analysis of two analytes by using an autonomous DNA machine that works in a two-cycle mode

We report a novel autonomous DNA machine for amplified electrochemical analysis of two DNAs. The DNA machine operates in a two‐cycle working mode to amplify DNA recognition events; the working mode is assisted by two different nicking endonucleases (NEases). Two bio‐barcode probes, a ZnS nanoparticle (NP)–DNA probe and a CdS NP–DNA probe, were used to trace two target DNAs. The detection system was based on a sensitive differential pulse anodic stripping voltammetry (DPASV) method for the simultaneous detection of Zn^II and Cd^II tracers, which were obtained by dissolving the two probes. Under the optimised conditions, detection limits as low as 5.6×10^?17 (3σ) and 4.1×10^?17 M (3σ) for the two target DNAs were achieved. It has been proven that the DNA machine system can simultaneously amplify two target DNAs by more than four orders of magnitude within 30 min at room temperature. In addition, in combination with an aptamer recognition strategy, the DNA machine was further used in the aptamer‐based amplification analysis of adenosine triphosphate (ATP) and lysozyme. With the amplification of the DNA machine, detection limits as low as 5.6×10^?9 M (3σ) for ATP and 5.2×10^?13 M (3σ) for lysozyme were simultaneously obtained. The satisfactory determination of ATP and lysozyme in Ramos cells reveals the good selectivity and feasibility of this protocol. The DNA machine is a promising tool for ultrasensitive and simultaneous multianalysis because of its remarkable signal amplification and simple machine‐like operation.     

Quantitation of Femtomolar‐Level Protein Biomarkers Using a Simple Microbubbling Digital Assay and Bright‐Field Smartphone Imaging

Quantitating ultra‐low concentrations of protein biomarkers is critical for early disease diagnosis and treatment. However, most current point‐of‐care (POC) assays are limited in sensitivity. Herein, we introduce an ultra‐sensitive and facile microbubbling assay for the quantification of protein biomarkers with a digital‐readout method that requires only a smartphone camera. We used machine learning to develop a smartphone application for automated image analysis to facilitate accurate and robust counting. Using this method, post‐prostatectomy surveillance of prostate specific antigen (PSA) can be achieved with a detection limit (LOD) of 2.1 fm (0.060 pg mL^?1), and early pregnancy detection using βhCG can be achieved with a of 0.034 mIU mL^?1 (2.84 pg mL^?1). This work provides the proof‐of‐principle of the microbubbling assay with a digital readout as an ultra‐sensitive technology with minimal requirement for power and accessories, facilitating future POC applications.     

Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5′-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

A single-molecule assay for multiple microRNA detection.     

Electrochemical Biosensors for Cancer Biomarkers Detection: Recent Advances and Challenges

Biomarkers are described as characteristics that provide information about biological conditions whether normal or pathological. Detection of biomarkers at the earliest stage of the cancer is of utmost importance for clinical diagnosis. Electrochemical biosensors allow detecting the low levels of specific analytes in blood, urine or saliva and providing a sensitive approach for direct measurement for cancer biomarker detection. Moreover, the integration of electrochemical devices with nanomaterials, such as carbon nanotubes, gold and magnetic particles offer amplification and multiplexing capabilities for simultaneous measurements of cancer biomarkers very sensitively. This review summarizes the recent developments of electrochemical biosensors systems for the detection of cancer biomarkers with emphasis on voltammetric, amperometric and impedimetric biosensors. A special attention is paid to aptamers and miRNAs that are very promising for the ultra‐sensitive and specific cancer biomarker detection.     

A novel method for the sensitive detection of mutant proteins using a covalent-bonding tube-based proximity ligation assay

Tumorigenesis is the cumulative result of multiple gene mutations. The mutant proteins that are expressed by mutant genes in cancer cells are secreted into the blood and are useful biomarkers for the early diagnosis of cancer. However, some difficulties exist; for example, the same gene will express different protein mutants in different patients, and early tumors secrete only small amounts of mutant protein. Thus, the presence of mutant proteins in plasma has not previously been exploited for the early diagnosis of cancer. Proximity ligation assay is a protein-detection method that has been developed in recent years and has been widely used because of its high sensitivity. However, this approach still suffers from some shortcomings that should be addressed. In this paper, we develop a covalent-bonding tube-based proximity ligation assay (TB-PLA). The limit of detection of TB-PLA for 0.001 pM, and the method exhibited a broad dynamic range of up to seven orders of magnitude. Furthermore, we coupled the conformation-specific antibody PAb240 of p53 mutants to PCR tubes for TB-PLA. The assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. Thus, we demonstrate TB-PLA to be a highly sensitive and effective approach that is suitable for the early clinical diagnosis of cancer using the conformation-specific antibodies of protein mutants.     

A HILIC‐MS/MS method for simultaneous quantification of the lysosomal disease markers galactosylsphingosine and glucosylsphingosine in mouse serum

Deficiencies of galactosylceramidase and glucocerebrosidase result in the accumulation of galactosylsphingosine (GalSph) and glucosylsphingosine (GluSph) in Krabbe and Gaucher diseases, respectively. GalSph and GluSph are useful biomarkers for both diagnosis and monitoring of treatment effects. We have developed and validated a sensitive, accurate, high‐throughput assay for simultaneous determination of the concentration of GalSph and GluSph in mouse serum. GalSph and GluSph and their deuterated internal standards were extracted by protein precipitation in quantitative recoveries, baseline separated by hydrophilic interaction chromatography and detected by positive‐ion electrospray mass spectrometry in multiple reaction monitoring mode. Total run time was 7 min. The lower limit of quantification was 0.2 ng/mL for both GalSph and GluSph. Sample stability, assay precision and accuracy, and method robustness were demonstrated. This method has been successfully applied to measurement of these lipid biomarkers in a natural history study in twitcher (Krabbe) mice.     

Surface Immobilization of Redox‐Labile Fluorescent Probes: Enabling Single‐Cell Co‐Profiling of Aerobic Glycolysis and Oncogenic Protein Signaling Activities

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface‐grafted resazurin molecules. The immobilization of the redox‐labile probes was achieved through chemical modifications on resazurin, followed by bio‐orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single‐cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug‐induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.     

Resistive-pulse DNA detection with a conical nanopore sensor

In this paper, we describe resistive-pulse sensing of two large DNAs, a single-stranded phage DNA (7250 bases) and a double-stranded plasmid DNA (6600 base pairs), using a conically shaped nanopore in a track-etched polycarbonate membrane as the sensing element. The conically shaped nanopore had a small-diameter (tip) opening of 40 nm and a large-diameter (base) opening of 1.5 microm. The DNAs were detected using the resistive-pulse, sometimes called stochastic sensing, method. This entails applying a transmembrane potential difference and monitoring the resulting ion current flowing through the nanopore. The phage DNA was driven electrophoretically through the nanopore (from tip to base), and these translocation events were observed as transient blocks in the ion current. We found that the frequency of these current-block events scales linearly with the concentration of the DNA and with the magnitude of the applied transmembrane potential. Increasing the applied transmembrane potential also led to a decrease in the duration of the current-block events. We also analyzed current-block events for the double-stranded plasmid DNA. However, because this DNA is too large to enter the tip opening of the nanopore, it could not translocate the pore. As a result, much shorter duration current-block events were observed, which we postulate are associated with bumping of the double-stranded DNA against the tip opening.     

Electrochemical Biosensing in Cancer Diagnostics and Follow‐up

In cancer, screening and early detection are critical for the success of the patient's treatment and to increase the survival rate. The development of analytical tools for non‐invasive detection, through the analysis of cancer biomarkers, is imperative for disease diagnosis, treatment and follow‐up. Tumour biomarkers refer to substances or processes that, in clinical settings, are indicative of the presence of cancer in the body. These biomarkers can be detected using biosensors, that, because of their fast, accurate and point of care applicability, are prominent alternatives to the traditional methods. Moreover, the constant innovations in the biosensing field improve the determination of normal and/or elevated levels of tumour biomarkers in patients’ biological fluids (such as serum, plasma, whole blood, urine, etc.). Although several biomarkers (DNA, RNA, proteins, cells) are known, the detection of proteins and circulating tumour cells (CTCs) are the most commonly reported due to their approval as tumour biomarkers by the specialized entities and commonly accepted for diagnosis by medical and clinical teams. Therefore, electrochemical immunosensors and cytosensors are vastly described in this review, because of their fast, simple and accurate detection, the low sample volumes required, and the excellent limits of detection obtained. The biosensing strategies reported for the six most commonly diagnosed cancers (lung, breast, colorectal, prostate, liver and stomach) are summarized and the distinct phases of the sensors’ constructions (surface modification, antibody immobilization, immunochemical interactions, detection approach) and applications are discussed.     

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.     

A Sandwich‐Type Electrochemical Biosensor for Detection of BCR/ABL Fusion Gene Using Locked Nucleic Acids on Gold Electrode

In this study, a sandwich‐type electrochemical enzyme‐based LNA‐modified DNA biosensor was developed to detect relative gene in chronic Myelogenous Leukemia first. This biosensor is based on a ‘sandwich’ detection strategy, which involves a pair of probes (a capture probe immobilized at the electrode surface and a reporter probe labeled biotin as an affinity tag for avidin‐HRP) modified LNA. Since biotin can be connected with avidin‐HRP, this biosensor offers an enzymatically amplified electrochemical current signal for the detection of target DNA. This new pattern exhibits high sensitivity and selectivity, and this biosensor has been used for an assay of PCR real sample with satisfactory result.     

Dual Amperometric Immunosensor for Improving Cancer Metastasis Detection by the Simultaneous Determination of Extracellular and Soluble Circulating Fraction of Emerging Metastatic Biomarkers

This paper reports the development of a dual immunosensor using magnetic microcarriers (MBs) and amperometric transduction at dual screen‐printed carbon electrodes (SPdCEs) for the simultaneous determination of two biomarkers: interleukin‐13 receptor α2 (IL‐13Rα2) and E‐cadherin (E‐CDH), with both extracellular and soluble fraction; oncogenic and tumor suppressor markers, respectively, of great relevance in metastatic processes. The implemented methodology involved the formation of sandwich‐type immunocomplexes using specific capture antibodies immobilized onto carboxylic acid magnetic microbeads (HOOC‐MBs), and biotinylated detector antibodies labeled with streptavidin?horseradish peroxidase conjugates (Strep‐HRP). The amperometric detection was performed by addition of hydrogen peroxide in the presence of hydroquinone (HQ) as the redox mediator. The dual immunosensing platform provided linear calibration ranges suitable for the determination of both biomarkers in liquid and solid clinical specimens as well as excellent selectivity against other cancer biomarkers. This simple handling dual bioplatform was applied to the determination of the soluble and extracellular fraction of the target biomarkers in serum and paraffined‐embedded tissues from colorectal cancer (CRC) patients diagnosed at different tumor grade. The obtained results reveal great potential of this configuration to improve the reliability in diagnosing metastatic CRC.     

Detection of single nucleotide polymorphisms by a gold nanowire-on-film SERS sensor coupled with S1 nuclease treatment

Single nucleotide polymorphisms (SNPs) can serve as important biomarkers for genetic diseases, for which accurate detection of SNPs is essential for early diagnosis. We have developed a novel SNP sensor by combining a Au nanowire-on-film surface-enhanced Raman scattering (SERS) platform with S1 nuclease reaction. The combined sensor system provides reproducible SERS signals only in the presence of perfectly matched target DNAs, to probe DNAs as a result of single-stranded DNA-specific degradation by S1 nuclease. Furthermore, point mutations in DNA causing Wilson disease and Avellino corneal dystrophy were successfully identified by this sensor, thereby indicating its practical ability to diagnose genetic diseases.     

Studies on the Affinity‐based Biosensors for Electrochemical Detection of HER2 Cancer Biomarker

As cancer diseases are the second main cause of death it is necessary to elaborate fast and efficient early diagnosis methods for their detection. One of the possibilities is the analysis of protein biomarkers, which abnormal concentration in physiological fluids might be an indication of cancer disease progression. Herein, we present the studies on the development of affinity‐based biosensors for electrochemical detection of HER2 protein, which is a common biomarker of breast cancer. The main objective was to verify the possibility of fabrication of HER2‐specific hybrid aptamer‐polyclonal antibody and antibody‐based sandwich sensing layer on gold electrode surface. The effectiveness of each electrode modification step was confirmed using voltammetric and impedimetric techniques in the presence of ferri/ferrocyanide redox couple. It was observed that hybrid construct was unlikely to be formed on the gold electrode due to a higher affinity of secondary polyclonal antibody towards target protein, which resulted in the separation of HER2‐antibody complex from the electrode surface. On the contrary, an antibody‐based sandwich receptor layer allowed for protein discrimination in the range from 1 to 100 ng mL^?1 by the application of TMB/H₂O₂ system and chronoamperometry detection technique. Though, the occurrence of interactions between interfering proteins and antibody‐based layer was noted, it led to at least two times smaller current responses than for HER2 protein.     

Magnetobiosensors Based on Viral Protein p19 for MicroRNA Determination in Cancer Cells and Tissues

MicroRNAs (miRs) have emerged as important clinical biomarkers with both diagnostic and prognostic value for relevant diseases, such as cancer. MiRs pose unique challenges for detection and are currently detected by northern blotting, real‐time PCR, and microarray techniques. These expensive, complicated, and time‐consuming techniques are not feasible for on‐site miR determination. In this study, amperometric magnetobiosensors involving RNA‐binding viral protein p19 as a selective biorecognition element were developed for miR quantification. The p19‐based magnetosensors were able to detect 0.4 fmol of a synthetic target and endogenous miR‐21 (selected as a model for its role in a wide variety of cancers) in only 2 h in total RNA extracted from cancer cells and human breast‐tumor specimens without PCR amplification and sample preprocessing. These results open up formidable perspectives for the diagnosis and prognosis of human cancers and for drug‐discovery programs.