Detection Of Multiple Tumor Markers Research Articles

A fluorescent platform integrated with a “one-pot” nicking endonuclease signal amplification and magnetic separation for simultaneous detection of tumor markers

Although Enzyme-linked immunosorbent assay (ELISA) has been widely used for biomedical research, simultaneous sensitive and cost-effective detection of multiple biomarkers is challenging. Herein, we proposed a “one-pot” nicking endonuclease signal amplification (NESA)-based fluorescent aptasensor for simultaneous detection of carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP). Firstly, two aptamers were synchronously immobilized on the surface of magnetic nanoparticles (MNPs) by coupling with two complementary DNA (cDNA). CEA and AFP specifically recognized the aptamers and then the released cDNA (ssDNA) from the double-strands (dsDNA) triggers NESA, further breaking two detection probes which were labeled with the fluorescent dye (FAM and ROX) and its quencher (BHQ1 and BHQ2) at the same time. Then, the fluorescence signal of FAM and ROX were restored separately. The results indicated that the fluorescence intensity at the emission wavelength of 518 nm and 610 nm had a positive correlation with CEA and AFP concentrations, respectively. Under the optimum conditions, wider liner range of 1–500 ng mL−1 to CEA and 5–800 ng mL−1 to AFP of this fluorescent aptasensor were successfully obtained, achieving a detection limit of CEA and AFP were 0.7 ng mL−1 and 2 ng mL−1, respectively. Hence, it turned out that the aptasensor strategy can be a promising candidate for developing a newly fluorescence assay for the simultaneous quantitative detection of multiple tumor markers in matrix samples by changing the corresponding sequences of aptamer and fluorescent signal probe, which has great potential for the screening of early cancer.

A Clinically Feasible Diagnostic Typing of Breast Cancer Built on a Homogeneous Electrochemical Biosensor for Simultaneous Multiplex Detection.

Simultaneous detection of multiple tumor markers is of great significance for an accurate diagnosis and early treatment of cancer. Electrochemical homogeneous biosensing strategies have been shown to have advantages, such as high sensitivity and no electrode modification, but they are still a challenge in the field of simultaneous detection of multiple tumor markers. The ER, PR, HER2, and Ki67 proteins are the standard biomarkers for the clinical molecular typing of breast cancer. Precise, sensitive, and simultaneous detection of these four biomarkers is of great importance in the molecular typing of breast cancer, which helps in the creation of personalized treatment plans. In the present study, we developed an electrochemical homogeneous electrochemical bioplatform based on metal ions/SiO2NPs/magnetic beads for detection of the four biomarkers and simultaneous diagnosis of the 10 types of breast cancer directly in human serum at one system by a single electrode. The electrochemical bioplatform has a short detection time of 140 min; however, the current clinical tissue testing time takes about 1 week. Also, the electrochemical bioplatform selectively detects HER2, ER, Ki67, and PR in a range of 0-1000 pg/mL with detection limits of 2, 1.8, 10.36, and 1.33 pg/mL, respectively.

Study on the application value of combined detection of multiple tumor markers in lung cancer classification diagnosis

Study on the application value of combined detection of multiple tumor markers in lung cancer classification diagnosis

A biothiols and H2O2 responsive fluorescence probe for selective cancer imaging

AbstractIdentification of cancer from normal tissues is important for early diagnosis of cancer. Combined detection of multiple tumor markers is important for accurate diagnosis. It is urgent to develop fluorescent probes that are responsive to multiple cancer characterizations for selective cancer imaging. Herein, we designed a novel near‐infrared (NIR) fluorescent probe (IRAPA) using a hemi‐cyanine skeleton as fluorophore and 3‐acrylamidopropanoic ester as recognizing unit that is responsive to both oxidative and reductive molecules. IRAPA has faint fluorescence emission as the intramolecular charge transfer (ICT) process is blocked. H2O2, glutathione (GSH) and cysteine (Cys) can individually induce the hydrolysis of ester bond and give fluorescent NIR IROH. IRAPA shows low cytotoxicity and produces strong fluorescence specifically in cancer cells/tissues. While the normal cells/tissues showed very weak fluorescence. Moreover, IRAPA shows higher differences between cancer and normal cells compared to probes that only response to biothiols or ROS.

Catalytic hairpin assembly-assisted dual-signal amplification platform for ultrasensitive detection of tumor markers and intelligent diagnosis of gastric cancer

Quantification of extracellular tumor markers has shown great promise for non-invasive cancer diagnosis. Combined detection of multiple tumor markers instead of a single one is valuable for accurate diagnosis. Here, we integrate CRISPR-Cas12a with DNA catalytic hairpin assembly (CHA) to doubly amplify the output signal for detecting microRNA-182 (miR-182), which is overexpressed by gastric cancer patients. Additionally, we develop a CHA system with self-replicating capacity (SRCHA) to realize dual-signal amplification for the detection of carcinoembryonic antigen (CEA), a broad-spectrum tumor marker. The proposed cascade amplification strategies enable ultrasensitive detection of miR-182 and CEA with low LODs of 0.063 fM and 4.8 pg mL−1, respectively. Moreover, we design a ternary “AND” logic gate using different concentrations of miR-182 and CEA as inputs, which demonstrates intelligent diagnosis of gastric cancer staging with a high accuracy of 93.3% in a clinical cohort of 30 individuals. Overall, our study expands the application of CRISPR-Cas12a in biosensing and provides a new diagnostic strategy for non-invasive liquid biopsy of gastric cancer before resorting to a traumatic tissue biopsy.

Electrochemical sensor for the simultaneous detection of CA72-4 and CA19-9 tumor markers using dual recognition via glycosyl imprinting and lectin-specific binding for accurate diagnosis of gastric cancer

The simultaneous identification and detection of multiple tumor markers provide more pathological information for the accurate diagnosis of cancer. In this study, a novel glycosyl imprinted sensor for the simultaneous detection of tumor markers CA19-9 and CA72-4 was prepared by combining the specific recognition of the glycosylated imprinting technique and lectin-characteristic glycan chains. The imprinted membrane was fabricated by electropolymerization using the characteristic glycopeptide STn on the surface of CA72-4 and the characteristic glycopeptide SLea on the surface of CA19-9 as template molecules and 2-aminophenylboronic acid as the functional monomer. To further improve the recognition efficiency, the specific binding of lectins to glycosyl chains was introduced. Sambucus nigra agglutinin I (SNA I) was labeled with cysteine, and Maackia amurensis lectin II (MAL II) was labeled with ferrocene. According to the specific binding of SNA to the Neu5Acα2-6GalNAcα-glycan chain in STn and MAL to α2, 3 sialic acid in SLea, CA72-4, and CA19-9 could be determined, respectively, after recombination between the glycosyl groups and GIP. The sensor shows high sensitivity to CA72-4 and CA19-9 in the concentration range of 0.005–200.0 U/mL, and it has been successfully applied to the detection of CA72-4 and CA19-9 in serum samples. The sensor provides a simple, fast, and low-cost alternative for accurate diagnosis of gastric cancer.

Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly.

Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag+ from the C-Ag+-C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag+, respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color- and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.

Association of multiple tumor markers with newly diagnosed gastric cancer patients: a retrospective study.

BackgroundThe purpose of this paper was to explore the correlation between multiple tumor markers and newly diagnosed gastric cancer.MethodsWe selected 268 newly diagnosed patients with gastric cancer and 209 healthy subjects for correlation research. The detection of multiple tumor markers was based on protein chips and the results were statistically analyzed using SPSS.ResultsWe concluded that gastric cancer was significantly related to gender, age, alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), carbohydrate antigen 199 (CA199), and carbohydrate antigen 242 (CA242) positive levels (P < 0.001). After CA199 and CA242 were stratified by gender, the male odds ratio (OR) was 30.400 and 31.242, respectively, while the female OR was 3.424. After CA125 was stratified by age in patients over 54 years old with gastric cancer, the risk of occurrence in the CA125-positive population was 16.673 times that of the CA125-negative patients. Among patients 54 years old and younger, being CA125-positive was not a risk factor for gastric cancer (P = 0.082). AFP, CEA, CA125, CA199, and CA242 positive levels during the M1 stage were statistically significant when compared with the M0 stage and control group (P < 0.001), but the AFP (P = 0.045) and CA125 (P = 0.752) positive levels were not statistically significant when compared with the M0 stage and control group. The combined detection sensitivity of multiple tumor markers was 44.78%.ConclusionOur research shows that gastric cancer is associated with age, gender, and the positive levels of AFP, CEA, CA125, CA199, and CA242. The positive levels of AFP and CA125 were related to the distant metastasis of gastric cancer. To a certain extent, the combined detection sensitivity can be used for the initial screening of gastric cancer.

Electrochemical Signal Substance for Multiplexed Immunosensing Interface Construction: A Mini Review.

Appropriate labeling method of signal substance is necessary for the construction of multiplexed electrochemical immunosensing interface to enhance the specificity for the diagnosis of cancer. So far, various electrochemical substances, including organic molecules, metal ions, metal nanoparticles, Prussian blue, and other methods for an electrochemical signal generation have been successfully applied in multiplexed biosensor designing. However, few works have been reported on the summary of electrochemical signal substance applied in constructing multiplexed immunosensing interface. Herein, according to the classification of labeled electrochemical signal substance, this review has summarized the recent state-of-art development for the designing of electrochemical immunosensing interface for simultaneous detection of multiple tumor markers. After that, the conclusion and prospects for future applications of electrochemical signal substances in multiplexed immunosensors are also discussed. The current review can provide a comprehensive summary of signal substance selection for workers researched in electrochemical sensors, and further, make contributions for the designing of multiplexed electrochemical immunosensing interface with well signal.

Value Analysis of Combined Detection of Multiple Tumor Markers in the Diagnosis and Treatment of Lung Cancer

Value Analysis of Combined Detection of Multiple Tumor Markers in the Diagnosis and Treatment of Lung Cancer

Simultaneous detection of CA15-3 and PGRMC1 on a microfluidic chip for early diagnosis of breast cancer

Simultaneous, rapid, and quantitative detection of multiple tumor markers is extremely important for the prevention and early diagnosis of cancers. In this study, we established a microfluidic-based indirect competitive chemiluminescence enzyme immunoassay for simultaneous detection of cancer antigen 15-3 (CA15-3) and progesterone receptor membrane components 1 (PGRMC1) for diagnosis of breast cancer. CA15-3 and PGRMC1 could be simultaneously detected on the chip. On the chip, the linear ranges of CA15-3 and PGRMC1 were 10 pg/mL–100 ng/mL and 20 pg/mL–358 ng/mL, respectively, whereas the limits of detection (LOD) of CA15-3 and PGRMC1 were 3 pg/mL and 5 pg/mL, respectively. Compared with the enzyme-linked immunosorbent assay (ELISA, LOD = 20 pg/mL for CA15-3; LOD = 10 pg/mL for PGRMC1), our method could simultaneously detect multiple samples with higher sensitivity, which could greatly improve the detection efficiency and meet the requirement for early diagnosis of breast cancer.

Highly sensitive electrochemical immunosensor for the simultaneous detection of multiple tumor markers for signal amplification

A novel sandwich-type electrochemical immunosensor has been fabricated for simultaneous determination of three kinds of tumor markers (TMs). The signal-amplified platform for sensor was assisted by graphene oxide (GO) loaded with Poly(styrene-alt-maleic anhydride) (PSMA). Three TMs of prostate specific antigen (PSA), human a-fetoprotein (AFP) and carbohydrate antigen 125 (CA 125) were employed as model test molecules. This sandwich-type immunoassay could detect the three antigens in differential pulse voltammetry (DPV) scan well at the same time. Under the optimized conditions, the multiplexed immunosensor displayed a splendid linear response in the range of 1.13 pg mL−1 - 113 ng mL−1 for PSA, 0.35 pg mL−1 - 35 ng mL−1 for AFP and 0.025 U mL−1 - 250 U mL−1 for CA 125. The detection limit was 86 fg mL−1, 14 fg mL−1 and 0.0019 U mL−1 for PSA, AFP and CA 125, respectively. This strategy provides a simple and sensitive method for immunoassay for the identification and validation of specific early cancers.

Dual-wavelength responsive photoelectrochemical aptasensor based on ionic liquid functionalized Zn-MOFs and noble metal nanoparticles for the simultaneous detection of multiple tumor markers.

A single tumor marker may correspond to a variety of diseases, and a specific disease requires the joint detection of multiple tumor markers for improving the accuracy of diagnoses. An ionic liquid-functionalized metal-organic framework (Zn-MOF microspheres) was used as the substrate to capture the aptamer (Ab1), and noble metal nanoparticles were used to label a signal aptamer (Ab2) to construct a dual-wavelength responsive sandwich-type photoelectrochemical (PEC) aptasensor. Due to the size effect, plasma resonance and the response of the noble metal nanoparticle enhancement system to different excitation wavelengths, the simultaneous detection of CEA and CA153 tumor markers was realized. Under the optimized conditions, CA153 and CEA at concentrations of 0.05-100 U mL-1 and 0.005-10 ng mL-1 were detected by the PEC aptasensor. Detection limits calculated for CA153 and CEA determinations were 0.0275 U mL-1 and 2.85 pg mL-1 (S/N = 3), respectively. CA153 and CEA in serum samples were detected by the PEC aptasensor, and their concentrations were well consistent with that obtained from the ELISA. In addition, the PEC aptasensor exhibited a recovery rate of 96.98%-103.4%, and a relative standard deviation of 1.1%-3.6%, indicating good practical value and accuracy, further confirming its potential for clinical diagnosis.

A sandwich-type electrochemical immunosensor for ultrasensitive detection of multiple tumor markers using an electrical signal difference strategy

Traditional sandwich-type electrochemical immunosensors can only detect single tumor markers because signal interference occurs when detecting multiple tumor markers. In this work, an electrical signal difference strategy was proposed for the accurate detection of multiple tumor markers. We labeled PdAgCeO2 mesoporous nanospheres with a carcinoembryonic antigen (CEA) secondary antibody and MnO2 nanosheets labeled with an alpha-fetoprotein (AFP) secondary antibody. The two electrical signal tags were mixed and incubated on a prepared immunosensor to catalyze H2O2 and generate an electrical signal I1 (i-t ampere curve). When 2.5 mM ascorbic acid solution (AA) was added to 20 mL of PBS solution at pH = 6.5 for 180 s, an electrical signal I2 was generated. I2 was the current response of the CEA antigen concentration, and the electrical signal difference ΔI = I1–I2 was the current response of the AFP antigen. Thus, the immunosensor accurately detected the AFP and CEA tumor markers. This method was called the electrical signal difference strategy. The proposed single-use immunosensor detected CEA antigens in a range of 0.001 ng/mL-40 ng/mL, and the detection limit was 0.5 pg/mL; the detection range of the AFP antigen was 0.005 ng/mL-100 ng/mL, and the detection limit was 1 pg/mL. Therefore, this study provides new ideas and strategies for accurate clinical detection of multiple tumor markers.

Salt-induced gold nanoparticles aggregation lights up fluorescence of DNA-silver nanoclusters to monitor dual cancer markers carcinoembryonic antigen and carbohydrate antigen 125

In clinical diagnosis of cancer, the monitoring of single tumor marker may result in many false and missed results, while simultaneous detection of multiple tumor markers should be more accuracy and effective. Here, we report a new strategy that salt-induced gold nanoparticles (AuNPs) aggregation lights up fluorescence of dual-color DNA-silver nanoclusters-aptamer (DNA-AgNCs-apta) for the simultaneous monitoring of carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA125). The dual-color aptasensor system is composed of green-emitting DNA-AgNCs with CEA aptamer (gDNA1-AgNCs-apta1) and red-emitting DNA-AgNCs with CA125 aptamer (rDNA2-AgNCs-apta2) in the ratio of 1:1 in volume. Upon addition of AuNPs, gDNA1-AgNCs-apta1 and/or rDNA2-AgNCs-apta2 are flexibly adsorbed onto the surface of AuNPs by terminal aptamer(s), which prevents salt-induced AuNPs aggregation under high salt condition and results in fluorescence quenching based on surface plasmon enhanced energy transfer (SPEET). With the addition of CEA and/or CA125, the target(s) and corresponding aptamer(s) coordinate to form the complex, keeping DNA-AgNCs-apta(s) far away from the surface of AuNPs and making AuNPs aggregated in high salt medium. The AuNPs aggregation leads to the recovery of fluorescence signals of DNA-AgNCs-apta(s) due to weakened SPEET. Utilizing the fluorescence aptasensor system, the limit of detection of CEA and CA125 are as low as 7.5 pg·mL-1 and 0.015 U·mL-1, respectively. The proposed method can be applied to the selective and simultaneous determination of CEA and CA125 in human serum.

Simultaneous Detection of Multiple Tumor Markers in Blood by Functional Liquid Crystal Sensors Assisted with Target-Induced Dissociation of Aptamer.

Multiplex detection of tumor markers in blood with high specificity and high sensitivity is critical to cancer diagnosis, treatment, and prognosis. Herein, we demonstrate a strategy for simultaneous detection of multiple tumor markers in blood by functional liquid crystal (LC) sensors assisted with target-induced dissociation (TID) of an aptamer for the first time. Magnetic beads (MBs) coated with an aptamer (apt1) are employed to specifically capture target proteins in blood. After incubation of the obtained protein-coated MBs with duplexes of another aptamer (apt2) and signal DNA, sandwich complexes of apt1/protein/apt2 are formed on the MBs due to specific recognition of target proteins by apt2, which induces release of signal DNA into the aqueous solution. Subsequently, signal DNA is specifically recognized by highly sensitive DNA-laden LC sensors. Using this strategy, a 3D printed optical cell was employed to enable simultaneous detection of multiple tumor markers such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and prostate specific antigen (PSA) with high specificity and high sensitivity. Overall, this effective and low-cost multiplex approach takes advantage of the easy separation of MBs, high specificity of aptamer-based recognition, and high sensitivity of functional LC sensors. Plus, it offers a performance that is competitive to that of commercial ELISA kits without potential interference from hemolysis, which makes it very promising in multiplex detection of tumor markers in clinical applications.

Simultaneous quantitative detection of multiple tumor markers in microfluidic nanoliter-volume droplets

We developed a simultaneous detection method for multiple tumor markers (TMs) in microfluidic droplets based on a multiple fluorescence resonance energy transfer (FRET) system. In this system, graphene oxide (GO) was used as the single quencher, while the multi-color quantum dots (QDs) labeled on different aptamers were employed as energy donors. When the aptamers were adsorbed onto GO due to the π-π stacking interaction, QDs were drawn to the surface of GO and quenched by it. Once the TMs were introduced, the corresponding fluorescence of QDs was recovered obviously owing to the preferential interaction of aptamers with the TMs. Here, the multi-FRET system was encapsulated into nanoliter-volume droplets by a simple T-junction microfluidic chip. The targets could be detected rapidly as the generated droplets flew through the integrated on-line detection zone. Three tumor markers, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF165) could be detected simultaneously in 33 nL-volume droplets, which is only 1/3000 of the volume of the sample consumed in the conventional fluorescence spectrophotometer. In addition, the signals corresponding to different TM targets in one nanoliter-volume droplet could be read out at the same time, and the signals could be output continuously owing to the uninterruptible generation of droplets. Even with a signal acquisition frequency of 55 droplets per minute, the multi-FRET biosensing system has linear ranges of 0.50–70 ng mL−1 for CEA, 0.25–70 ng mL−1 for PSA and 0.50–70 ng mL−1 for VEGF165. The detection limits of CEA, PSA and VEGF165 were calculated to be 0.15 ng mL−1, 0.035 ng mL−1 and 0.11 ng mL−1, respectively. The method was also validated by analyzing human serum sample dilutions. The proposed multi-FRET-based system has potential to become a powerful tool for rapid, low-cost and simultaneous detection of multiple tumor markers.

Multiplex measurement of twelve tumor markers using a GMR multi-biomarker immunoassay biosensor

Tumor markers play an important role in the early diagnosis and therapeutic effect monitoring of tumors. Combined detection of multiple tumor markers is a realistic way of improving the sensitivity and specificity of cancer diagnosis. To achieve this, we studied and designed a giant magneto resistance (GMR) multi-biomarker immunoassay biosensor that can simultaneously detect twelve kinds of tumor markers by integrating a GMR sensor chip, a microfluidic device, a magnetic nano-beads label, and a double antibody sandwich immunoassay method. As a proof of concept, the proposed immunosensor was utilized to detect 12 tumor markers (AFP, CEA, CYFRA21-1, NSE, SCC, PG I, PG II, CA19-9, total PSA, free PSA, free-β-hCG, Tg) and to screen patients with lung cancer, liver cancer, digestive tract cancer, prostatic cancer, etc. The immunosensor showed excellent sensitivity, accuracy, precision and stability. Designed as a POCT device, the immunosensor also allows for portability, able to perform rapid detection wherever necessary. As a multi-analyte assay, it provides significant advantages over single-analyte tests in terms of cost per test, labor and convenience. The system's ability to simultaneously measure the concentration of multiple markers in serum samples with excellent sensitivity and accuracy allows the immunosensor to be used for early tumor diagnosis.

SERS-based multiplex immunoassay of tumor markers using double SiO2@Ag immune probes and gold-film hemisphere array immune substrate

Highly sensitive and specific detection of tumor markers is extremely significant for the early diagnosis and treatment of cancer. We proposed a novel surface-enhanced Raman scattering (SERS)-based multiplex immunoassay proposal to implement the ultrasensitive detection of multiple tumor markers. In our work, the prostate specific antigen (PSA) and α-fetoprotein (AFP) as two kinds of target analytes were simultaneously detected by using the sandwich immune complex consisted of 4MBA-labeled SiO2@Ag immune probes, 4NTP-labeled SiO2@Ag immune probes and the gold-film hemisphere array (Au-FHA) immune substrate. And the selection of Raman molecules and the regulation of SERS signals among the probes are the technical keys to realize the effective multiplex immunoassay. The experiment results demonstrate that the constructed immunoassay platform exhibits a wider dynamic linear range from 10 fg mL−1 to 400 ng mL−1 and the limit of detection of 3.38 and 4.87 fg mL−1 for PSA and AFP, respectively. And more, the CA-125 was selected as an unspecific antigen to verify the high-specificity of the multiplex immunoassay. In addition, the PSA and AFP in human serum samples had been detected by the proposed immunoassay proposal and the test data present a good consistent with that of chemiluminescent immunoassay (CLIA). It is expected that the SERS-based multiplex immunoassay proposal could be applied in the practical clinical diagnoses of cancer.

Simultaneous detection of multiple tumor markers by label-free electrochemical immunoassay using chip-like glass carbon electrodes

Simultaneous detection of multiple tumor markers by label-free electrochemical immunoassay using chip-like glass carbon electrodes