Real-time prostate-specific antigen detection with prostate-specific antigen imprinted capacitive biosensors

Prostate specific antigen (PSA) is a valuable biomarker for early detection of prostate cancer, the third most common cancer in men. Ultrasensitive detection of PSA is crucial to screen the prostate cancer in an early stage and to detect the recurrence of the disease after treatment. In this report, microcontact-PSA imprinted (PSA-MIP) capacitive biosensor chip was developed for real-time, highly sensitive and selective detection of PSA. PSA-MIP electrodes were prepared in the presence of methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker via UV polymerization. Immobilized Anti-PSA antibodies on electrodes (Anti-PSA) for capacitance measurements were also prepared to compare the detection performances of both methods. The electrodes were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV) and real-time PSA detection was performed with standard PSA solutions in the concentration range of 10 fg mL⁻¹–100 ng mL⁻¹. The detection limits were found as 8.0 × 10⁻⁵ ng mL⁻¹ (16 × 10⁻¹⁷ M) and 6.0 × 10⁻⁴ ng mL⁻¹ (12 × 10⁻¹⁶ M) for PSA-MIP and Anti-PSA electrodes, respectively. Selectivity studies were performed against HSA and IgG and selectivity coefficients were calculated. PSA detection was also carried out from diluted human serum samples and finally, reproducibility of the electrodes was tested. The results are promising and show that when the sensitivity of the capacitive system is combined with the selectivity and reproducibility of the microcontact-imprinting procedure, the resulting system might be used successfully for real-time detection of various analytes even in very low concentrations.     

Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization

A simple but highly sensitive colorimetric method was developed to detect cancer cells based on aptamer–cell interaction. Cancer cells were able to capture nucleolin aptamers (AS 1411) through affinity interaction between AS 1411 and nucleolin receptors that are over expressed in cancer cells, The specific binding of AS 1411 to the target cells triggered the removal of aptamers from the solution. Therefore no aptamer remained in the solution to hybridize with complementary ssDNA-AuNP probes as a result the solution color is red. In the absence of target cells or the presence of normal cells, ssDNA-AuNP probes and aptamers were coexisted in solution and the aptamers assembled DNA-AuNPs, produced a purple solution. UV–vis spectrometry demonstrated that this hybridization-based method exhibited selective colorimetric responses to the presence or absence of target cells, which is detectable with naked eye. The linear response for MCF-7 cells in a concentration range from 10 to 10⁵ cells was obtained with a detection limit of 10 cells. The proposed method could be extended to detect other cells and showed potential applications in cancer cell detection and early cancer diagnosis.     

Colorimetric detection of hepatitis B virus (HBV) DNA based on DNA-templated copper nanoclusters

DNA detection plays an important role in early diagnosis of genetic disease. The conventional detection methods of DNA are based on expensive equipment, which do not meet the demands of developing countries. Thus, we developed a colorimetric method, which could be observed with naked eye and used copper nanoclusters for cost-effective. Moreover, the target of this method is the DNA in Hepatitis B virus that is one of the most popular chronic viral infections in developing countries over the past years. Our method was sensitive and the limit of detection was 12 × 10⁹ molecules. Three-base-pair mismatches target DNA was detected easily. These results revealed the favorable sensitivity and selectivity of this approach. Most importantly, our method may have potential applications in correct diagnosis of genetic disease and monitoring of gene therapy in the poverty-stricken areas.     

Design of a dual aptamer-based recognition strategy for human matrix metalloproteinase 9 protein by piezoelectric biosensors

MMP-9, human matrix metalloproteinase 9, belongs to the family of zinc-dependent peptide-bond hydrolases and is involved in the degradation of the extracellular matrix (ECM). In clinics, it is well known that elevated MMP-9 serum levels are associated with cardiovascular dysfunctions, several aspects of the physiology and pathology of the central nervous system, neuropsychiatric disorders and degenerative diseases related to brain tumors, and excitotoxic/neuroinflammatory processes. Due to the large interest of diagnostics in this protein, efforts to set up sensitive methods to detect MMP-9 for early diagnosis of a number of metabolic alterations are rapidly increasing. In this panorama, biosensors could play a key role; therefore we explored for the first time the development of an aptamer-based piezoelectric biosensor for a sensitive, label free, and real time detection of MMP-9. The detecting strategy involved two different aptamers in a sandwich-like approach able to detect down to 100 pg mL⁻¹ (1.2 pM) of MMP-9 as detection limit in standard solution. As proof of principle, commercial serum was investigated in terms of possible interferents, their identification and role in MMP-9 detection. The estimated detection limit for MMP-9 is about 560 pg mL⁻¹ (6.8 pM) in untreated serum.     

CH-overtone regions as diagnostic markers for near-infrared spectroscopic diagnosis of primary cancers in human pancreas and colorectal tissue

We have investigated the application of near-infrared spectroscopy for detection of human primary pancreatic and colorectal cancers. Spectra from cancerous and normal tissue were collected from a total of 37 surgically resected pancreatic and colorectal patient tissue specimens using a fibre-optic probe. Major spectral differences were observed in the CH-stretching first (6,000–5,400 cm⁻¹) and second overtone (9,000–7,900 cm⁻¹) regions. By use of artificial neural networks, linear discriminant analysis, and cluster analysis as pattern-recognition methods the spectra were classified into cancerous and normal tissue groups with accuracy up to 89%. We also explored differences between the spectra obtained from colorectal and pancreatic tissue. Spectral data from cancerous and normal tissue were classified organ-specifically into four groups with accuracy between 80 and 83%. Our results indicate that CH-overtone regions, besides serving as diagnostic markers for NIR spectroscopic diagnosis of primary human pancreas and colorectal cancers, are also useful for elucidating differences between the spectra obtained from colorectal and pancreatic cancerous tissue.     

Multianalyte immunoassay chip for detection of tumor markers by chemiluminescent and colorimetric methods

Most cancers developed an elevation of the level of at least two markers associated with their incidence. Simultaneous detection of multi-tumor markers associated with a particular type of cancer plays an important role in cancer diagnostic. Here, a multianalyte immunoassay chip for simple and sensitive detection of tumor markers with chemiluminescent and colorimetric methods was proposed, in which carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) that associated with colorectal cancer were detected as model. The immunoassay chip was fabricated by co-immobilization of CEA/CA19-9 antibody on a glass slide with γ-glycidoxypropyltrimethoxysilane as linkage. Through sandwiched immunoreactions, CEA, CA19-9, and their corresponding enzyme tracers, alkaline phosphatase-labeled anti-CEA and horseradish peroxidase-labeled anti-CA19-9, were introduced on the chip. Then, they were sequentially detected by chemiluminescent method in the range of 0.5–80 μg/L and 0.5–80 kU/L with the detection limits of 0.41 μg/L and 0.36 kU/L at 3σ for CEA and CA19-9, respectively. They could also be detected by colorimetric method in the range of 1–200 μg/L and 5–200 kU/L with the detection limits of 0.25 μg/L and 1.25 kU/L at 3σ for CEA and CA19-9, respectively. All these results demonstrated that the present work provided a promising analytical method for tumor markers’ analysis with the advantages of simple analytical procedure, small sample volume and lower cost, which made the proposed method potential for high-throughput detection.     

A Ti3C2TX/Pt–Pd based amperometric biosensor for sensitive cancer biomarker detection

The detection of cancer biomarkers is of great significance for the early screening of cancer. Detecting the content of sarcosine in blood or urine has been considered to provide a basis for the diagnosis of prostate cancer. However, it still lacks simple, high-precision and wide-ranging sarcosine detection methods. In this work, a Ti₃C₂T_X/Pt–Pd nanocomposite with high stability and excellent electrochemical performance has been synthesized by a facile one-step alcohol reduction and then used on a glassy carbon electrode (GCE) with sarcosine oxidase (SO_x) to form a sarcosine biosensor (GCE/Ti₃C₂T_X/Pt–Pd/SO_x). The prominent electrocatalytic activity and biocompatibility of Ti₃C₂T_X/Pt–Pd enable the SO_x to be highly active and sensitive to sarcosine. Under the optimized conditions, the prepared biosensor has a wide linear detection range to sarcosine from 1 to 1000 µM with a low limit of detection of 0.16 µM (S/N = 3) and a sensitivity of 84.1 µA/mM cm². Besides, the reliable response in serum samples shows its potential in the early diagnosis of prostate cancer. More importantly, the successful construction and application of the amperometric biosensor based on Ti₃C₂T_X/Pt–Pd will provide a meaningful reference for detecting other cancer biomarkers.     

Horseradish peroxidase and aptamer dual-functionalized nanoprobe for the amplification detection of alpha-methylacyl-CoA racemase

Alpha-methylacyl-CoA racemase (AMACR) is over-expressed in many cancer types and can serve as a novel diagnostic biomarker. Development of convenient and sensitive detection methods of AMACR is of particular importance for cancer diagnosis. Aptamers are a type of recognition elements, which possess many advantages over antibody, making them suitable for applications in biosensing and biotechnology. In this work, we use the efficient surface modification of gold nanoparticles (AuNPs) to prepare the horseradish peroxidase (HRP) and aptamer dual-functionalized nanoprobe. The immobilization of HRP and thiol-terminated aptamer on the surface of AuNPs can be achieved through electrostatic interaction and the formation of Au–S bond, respectively. This nanoprobe, which is used as discriminating and catalytic probe, can be combined with enzyme immunoassay method to increase the detection sensitivity of AMACR. The detection limit can reach as low as 4.6 pg mL⁻¹ due to the dual signal amplification from enzymatic cycling and the high loading of enzymes on AuNPs. This sensitivity is about three orders of magnitude higher than that of AMACR aptamer based fluorescence method, which is also comparable to or one order of magnitude higher than that of ELISA. Furthermore, this method is more simple and effective, which not only avoids the conjugation between recognition element and the catalytic enzyme, but also achieves greater signal amplification. This assay could be used as a sensitive and selective platform for the detection of target protein.     

Early Diagnosis of Colorectal Cancer in Rats With DMH Induced Carcinogenesis by Means of Urine Autofluorescence Analysis

Cancer is one of the most highlighted topics of current research. Early detection of this disease allows more effective therapy, hence higher chance of cure. Application of fluorescence spectral techniques into oncological diagnostic is one of the potential alternatives. Chemically induced carcinogenesis in rats is widely used model for exploration of various aspects of colorectal cancer. This study shows value of discriminate analysis of urine fluorescent fingerprint between healthy control group of rats and those with dimethylhydrazine induced early lesions of colorectal cancer. Using fluorescence spectroscopy, significant difference (P < 0.05) between both of group was achieved.     

Pulsed amperometric detection at glassy carbon electrodes: A new waveform for sensitive and reproducible determination of electroactive compounds

In this work, the application of a new pulsed amperometric detection (PAD) waveform at a glassy carbon electrode, operating in typical chromatographic mobile phases, is proposed for the sensitive and reproducible determination of arylethanolaminic and phenolic moiety based compounds (e.g. beta-agonists and polyphenols). Preliminary experiments by cyclic voltammetry were carried out to investigate the electrochemical behaviour and to select the detection and cleaning electrode potentials. The proposed potential-time profile was designed to prevent the carbon electrode fouling under repeated analyses, thus ensuring a reproducible and sensitive quantitative determination, without the need of any mechanical or chemical electrode cleaning procedure. The waveform electrochemical parameters, including detection and delay times, were optimized in terms of sensitivity, limit of detection and response stability. The optimized waveform allowed the sensitive and stable detection of model compounds, such as clenbuterol and caffeic acid, that showed detection limits of 0.1 μg L⁻¹ and 14 μg L⁻¹, quantification limits of 0.4 μg L⁻¹ and 46 μg L⁻¹, and linearity up to 100 μg L⁻¹ (r = 0.9993) and 10 mg L⁻¹ (r = 0.9998), respectively. Similar results were obtained for other compounds of the same classes, with precision values under repeatability conditions ranging from 3.0 to 5.9%. The proposed method can be then considered as an excellent alternative to the post-column detection of beta-agonists, phenols and polyphenols.     

Sensitive detection of human breast cancer cells based on aptamer–cell–aptamer sandwich architecture

Breast cancer is one of the most critical threats to the health of women, and the development of new methods for early diagnosis is urgently required, so this paper reports a method to detect Michigan cancer foundation-7 (MCF-7) human breast cancer cells with considerable sensitivity and selectivity by using electrochemical technique. In this method, a mucin 1 (MUC1)-binding aptamer is adopted to recognize MCF-7 human breast cancer cells, while enzyme labeling is employed to produce amplified catalytic signals. The molecular recognition and the signal amplification are elaborately integrated by fabricating an aptamer–cell–aptamer sandwich architecture on an electrode surface, thus a biosensor for the detection of MCF-7 is fabricated based on the architecture. The detection range can be from 100 to 1 × 10⁷ cells, and the detection limit can be as low as 100 cells. The method is also cost-effective and conveniently operated, implying potential help for the development of early diagnosis of breast cancer.     

Fabrication of highly catalytic silver nanoclusters/graphene oxide nanocomposite as nanotag for sensitive electrochemical immunoassay

Silver nanoclusters and graphene oxide nanocomposite (AgNCs/GRO) is synthesized and functionalized with detection antibody for highly sensitive electrochemical sensing of carcinoembryonic antigen (CEA), a model tumor marker involved in many cancers. AgNCs with large surface area and abundant amount of low-coordinated sites are synthesized with DNA as template and exhibit high catalytic activity towards the electrochemical reduction of H₂O₂. GRO is employed to assemble with AgNCs because it has large specific surface area, super electronic conductivity and strong π-π stacking interaction with the hydrophobic bases of DNA, which can further improve the catalytic ability of the AgNCs. Using AgNCs/GRO as signal amplification tag, an enzyme-free electrochemical immunosensing protocol is designed for the highly sensitive detection of CEA on the capture antibody functionalized immunosensing interface. Under optimal conditions, the designed immunosensor exhibits a wide linear range from 0.1 pg mL⁻¹ to 100 ng mL⁻¹ and a low limit of detection of 0.037 pg mL⁻¹. Practical sample analysis reveals the sensor has good accuracy and reproducibility, indicating the great application prospective of the AgNCs/GRO in fabricating highly sensitive immunosensors, which can be extended to the detection of various kinds of low abundance disease related proteins.     

Nanogold-penetrated poly(amidoamine) dendrimer for enzyme-free electrochemical immunoassay of cardiac biomarker using cathodic stripping voltammetric method

Methods based on immunoassays have been developed for cardiac biomarkers, but most involve the low sensitivity and are unsuitable for early disease diagnosis. Herein we design an electrochemical immunoassay for sensitive detection of myoglobin (a cardiac biomarker for acute myocardial infarction) by using nanogold-penetrated poly(amidoamine) dendrimer (AuNP-PAMAM) for signal amplification without the need of natural enzymes. The assay was carried out on the monoclonal mouse anti-myoglobin (capture) antibody-anchored glassy carbon electrode using polyclonal rabbit anti-myoglobin (detection) antibody-labeled AuNP-PAMAM as the signal tag. In the presence of target myoglobin, the sandwiched immunocomplex could be formed between capture antibody and detection antibody. Accompanying AuNP-PAMAM, the carried gold nanoparticles could be directly determined via stripping voltammetric method under acidic conditions. Under optimal conditions, the detectable electrochemical signal increased with the increasing target myoglobin in the sample within a dynamic working range from 0.01 to 500 ng mL⁻¹ with a detection limit of 3.8 pg mL⁻¹. The electrochemical immunoassay also exhibited high specificity and good precision toward target myoglobin. Importantly, our strategy could be applied for quantitative monitoring of myoglobin in human serum specimens, giving well matched results with those obtained from commercialized enzyme-linked immunosorbent assay (ELISA) method.     

Rapid identification and high sensitive detection of cancer cells on the gold nanoparticle interface by combined contact angle and electrochemical measurements

In this study, we have proposed a novel strategy for the rapid identification and high sensitive detection of different kinds of cancer cells by means of electrochemical and contact angle measurements. A simple, unlabeled method based on the functionalized Au nanoparticles (GNPs) modified interface has been utilized to distinguish the different cancer cells, including lung cancer cells, liver cancer cells, drug sensitive leukemia K562/B.W cells and drug resistant leukemia K562/ADM cells. The relevant results indicate that under optimal conditions, this method can provide the quantitative determination of cancer cells, with a detection limit of ∼10³ cells mL⁻¹. Our observations demonstrate that the difference in the hydrophilic properties for target cellular surfaces and in the uptake efficiency of the anticancer drug daunorubicin for different cancer cells could be readily chosen as the elements of cancer identification and sensitive detection. This raises the possibility to advance the promising clinic diagnosis and monitoring of tumors with the aim of successful chemotherapy of human cancers.     

An ultrasensitive electrochemical immunosensor for CA72-4 based on a signal amplification strategy of MoS2 nanoflower-supported Au nanoparticles

Accurate detection of cancer antigen 72-4 (CA72-4), a tumor-associated glycoprotein, is of great significance for gastric cancer diagnosis and immunotherapy monitoring. Modification of noble metal nanoparticles on transition metal dichalcogenides can significantly enhance functions, such as electron transport. Molybdenum disulfide gold nanoparticles nanocomposites (MoS₂-Au NPs) were prepared in this study and a series of characterization studies were carried out. In addition, a label-free, highly sensitive electrochemical immunosensor molybdenum disulfide -Au nanoparticles/Glassy carbon electrode (MoS₂-Au NPs/GCE) was also prepared and used for the detection of CA72-4. The electrochemical performance of the immunosensor was characterized by electrochemical techniques, such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The results indicated that better MoS₂-Au NPs nanomaterials have been synthesized, and the prepared electrochemical immunosensor, MoS₂-Au NPs/GCE, showed excellent electrochemical performance. The sensor exhibited high detection sensitivity under optimal conditions, including an incubation time of 30 min, an incubation temperature of 25 °C, and a pH of 7.0. The electrochemical immunosensor also had a low detection limit of 2.0 × 10^?5 U/mL (S/N = 3) in a concentration range of 0.001–200 U/mL, with good selectivity, stability, and repeatability. In conclusion, this study provided a theoretical basis for the highly sensitive detection of tumor markers in clinical biological samples.     

Solubilization of Hexyl Aminolevulinate by Surfactants for Tumor Fluorescence Detection

Hexyl aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinic acid (5-ALA) and can induce more protoporphyrin IX (PpIX) formation and stronger fluorescence intensity (FI) than 5-ALA, which will greatly facilitate photodynamic diagnosis and therapy. The main drawback of HAL is its low solubility in neutral aqueous media. In this study, surfactants were used to increase HAL solubility in the cell culture medium and serum, followed by in vitro fluorescence formation measurement in human pancreatic cancer cells (SW1990) and in vivo fluorescence detection in tumor-bearing mice. The results showed that Tween 80 (TW80) and Kolliphor® HS 15 (HS15) increased the solubility of HAL in the selected media. Although TW80 and HS15 exhibited in vitro cytotoxicity at high concentrations (5 mg mL⁻¹), they facilitated fluorescent signal formation at the early stage of cell incubation. When surfactants were used, the FI should be determined without the routine washing process because surfactant-containing culture medium caused the loss of synthesized PpIX during the washing process. When HAL dissolved in TW80 solution was injected intraperitoneally into pancreatic cancer-bearing mice at a dose of 50 mg kg⁻¹, the tumors exhibited red fluorescence, which indicated that systemic administration of surfactant-solubilized HAL might be applicable for tumor fluorescence detection in vivo.     

Coupling hybridization chain reaction with DNAzyme recycling for enzyme-free and dual amplified sensitive fluorescent detection of methyltransferase activity

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg²⁺-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg²⁺ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10⁻⁴ U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.     

Multifunctional nanoprobe for cancer cell targeting and simultaneous fluorescence/magnetic resonance imaging

Multifunctional nanoprobes with distinctive magnetic and fluorescent properties are highly useful in accurate and early cancer diagnosis. In this study, nanoparticles of Fe₃O₄ core with fluorescent SiO₂ shell (MFS) are synthesized by a facile improved Stöber method. These nanoparticles owning a significant core-shell structure exhibit good dispersion, stable fluorescence, low cytotoxicity and excellent biocompatibility. TLS11a aptamer (Apt1), a specific membrane protein for human liver cancer cells which could be internalized into cells, is conjugated to the MFS nanoparticles through the formation of amide bond working as a target-specific moiety. The attached TLS11a aptamers on nanoparticles are very stable and can't be hydrolyzed by DNA hydrolytic enzyme in vivo. Both fluorescence and magnetic resonance imaging show significant uptake of aptamer conjugated nanoprobe by HepG2 cells compared to 4T1, SGC-7901 and MCF-7 cells. In addition, with the increasing concentration of the nanoprobe, T₂-weighted MRI images of the as-treated HepG2 cells are significantly negatively enhanced, indicating that a high magnetic field gradient is generated by MFS-Apt1 which has been specifically captured by HepG2 cells. The relaxivity of nanoprobe is calculated to be 11.5 mg⁻¹s⁻¹. The MR imaging of tumor-bearing nude mouse is also confirmed. The proposed multifunctional nanoprobe with the size of sub-100 nm has the potential to provide real-time imaging in early liver cancer cell diagnosis.