High Sensitive Detection of Prostate Specific Antigen by Using Ferrocene Cored Asymmetric PAMAM Dendrimer Interface Screen Printed Electrodes

In this study, electrochemical immunosensors were developed for the detection of prostate specific antigen (PSA) using ferrocene (Fc) and polyamidoamine dendrimer (PAMAM) constructs. The biosensor fabrication was designed by modifying the screen‐printed gold electrode (Au) with ferrocene cored dendrimers (FcPAMAM) synthesized in three different generations. The self‐assembled monolayer principle was followed, to obtain sensitive, selective and disposable electrodes. Therefore, the Au electrodes were modified with cysteamine (Cys) to obtain a functional surface for FcPAMAM dendrimers to bind. Dendrimer generations were attached to this surface using a cross‐linker (glutaraldehyde) so that a suitable surface was obtained for binding of biological components. The Monoclonal PSA antibody (anti‐PSA) was immobilized on the Au electrode surface which coated with dendrimer, and (Au/Cys/FcPAMAM/anti‐PSA) biosensing electrode was obtained. The PSA detection performances of electrochemical impedance spectroscopy (EIS) and Amperometry based immunosensors exhibited very low detection limits; 0.001 ng mL^?1 and 0.1 pg mL^?1, respectively. In addition, EIS and Amperometry based biosensors using Au/Cys/FcPAMAM/anti‐PSA sensing electrode were represented excellent linear ranges of 0.01 ng mL^?1 to 100 ng mL^?1 and 0.001 ng mL^?1 to 100 ng mL^?1. In order to determine the applicability recovery and selectivity tests were performed using three different proteins in human serum.     

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.     

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 10² to 1 × 10⁷ cells mL⁻¹ and high sensitivity with a low detection limit of 30 cells mL⁻¹. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.     

Synergistic Roles of Curcumin in Sensitising the Cisplatin Effect on a Cancer Stem Cell-Like Population Derived from Non-Small Cell Lung Cancer Cell Lines

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.     

Fabrication of immunosensor microwell arrays from gold compact discs for detection of cancer biomarker proteins

A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm(2) with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL(-1) (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL(-1). These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.     

CD133 and CD133‐regulated nucleophosmin linked to 5‐fluorouracil susceptibility in human colon cancer cell line SW620

Cancer stem cells (CSCs) are known to be resistant to conventional chemotherapy and radiotherapy. Specific CSC targeting and eradication is therefore a therapeutically important challenge. CD133 is a colorectal CSC marker with unknown function(s). Assessing proteomic changes induced by CD133 may provide clues not only to new CD133 functions but also to the chemotherapy and radiation susceptibility of colon cancer cells. To identify the proteins affected by CD133, CD133‐positive (CD133+), and CD133‐negative (CD133–) human colon cancer cells were obtained by cell sorting. Whole proteomes were profiled from SW620/CD133+ and SW620/CD133– cells and analyzed by 2D‐based proteome analysis. Nucleophosmin (NPM1) was identified as a protein regulated by CD133. CD133 protein level was not affected by NPM1, and an interaction between the two proteins was not observed. CD133 and NPM1 protein levels were positively correlated in 11 human colon cancer cell lines. The CD133+ subpopulation percentage or its value normalized against CD133 protein level was only linked to intrinsic susceptibility of human colon cancer cells to 5‐fluorouracil (5‐FU). However, either suppression of CD133 or NPM1 significantly increased 5‐FU susceptibility of SW620. The present study suggests that CD133‐regulated NPM1 protein level may provide a clue to novel CD133 function(s) linked to human colon cancer cell susceptibility to chemotherapy.     

Molecular docking study of natural alkaloids as multi-targeted hedgehog pathway inhibitors in cancer stem cell therapy

Cancer is responsible for millions of deaths throughout the world every year. Increased understanding as well as advancements in the therapeutic aspect seems suboptimal to restrict the huge deaths associated with cancer. The major cause responsible for this is high resistance as well as relapse rate associated with cancers. Several evidences indicated that cancer stem cells (CSC) are mainly responsible for the resistance and relapses associated with cancer. Furthermore, agents targeting a single protein seem to have higher chances of resistance than multitargeting drugs. According to the concept of network model, partial inhibition of multiple targets is more productive than single hit agents. Thus, by fusing both the premises that CSC and single hit anticancer drugs, both are responsible for cancer related resistances and screened alkaloids for the search of leads having CSC targeting ability as well as the capability to modulating multiple target proteins. The in silico experimental data indicated that emetine and cortistatin have the ability to modulate hedgehog (Hh) pathway by binding to sonic hedgehog (Hh), smoothened (Smo) and Gli protein, involved in maintenance CSCs. Furthermore, solamargine, solasonine and tylophorine are also seems to be good lead molecules targeting towards CSCs by modulating Hh pathway. Except solamargine and solasonine, other best lead molecules also showed acceptable in silico ADME profile. The predicted lead molecules can be suitably modified to get multitargeting CSC targeting agent to get rid of associate resistances.     

A Copper(II) Phenanthroline Metallopeptide That Targets and Disrupts Mitochondrial Function in Breast Cancer Stem Cells

The breast cancer stem cell (CSC) and bulk breast cancer cell potency of a series of metallopeptides containing dichloro(1,10‐phenanthroline)copper(II) and various organelle‐targeting peptide sequences is reported. The mitochondria‐targeting metallopeptide 1 exploits the higher mitochondrial load in breast CSCs over the corresponding non‐CSCs and the vulnerability of breast CSCs to mitochondrial damage to potently and selectively kill breast CSCs. Strikingly, 1 reduces the formation and size of mammospheres to a greater extent than salinomycin, an established CSC‐potent agent. Mechanistic studies show that 1 enters CSC mitochondria, induces mitochondrial dysfunction, generates reactive oxygen species (ROS), activates JNK and p38 pathways, and prompts apoptosis. To the best of our knowledge, 1 is the first metallopeptide to selectivity kill breast CSCs in vitro.     

Breast Cancer Stem Cell Potent Copper(II)–Non‐Steroidal Anti‐Inflammatory Drug Complexes

The breast cancer stem cell (CSC) potency of a series of copper(II)–phenanthroline complexes containing the nonsteroidal anti‐inflammatory drug (NSAID), indomethacin, is reported. The most effective copper(II) complex in this series, 4 , selectivity kills breast CSC‐enriched HMLER‐shEcad cells over breast CSC‐depleted HMLER cells. Furthermore, 4 reduces the formation, size, and viability of mammospheres, to a greater extent than salinomycin, a potassium ionophore known to selectively inhibit CSCs. Mechanistic studies revealed that the CSC‐specificity observed for 4 arises from its ability to generate intracellular reactive oxygen species (ROS) and inhibit cyclooxygenase‐2 (COX‐2), an enzyme that is overexpressed in breast CSCs. The former induces DNA damage, activates JNK and p38 pathways, and leads to apoptosis.     

A disposable immunosensor based on gold colloid modified chitosan nanoparticles-entrapped carbon paste electrode

A new strategy is described to construct disposable electrochemical immunosensors for the assay of human immunoglobulin. It is based on a carbon paste electrode constructed from chitosan nanoparticles modified with colloidal gold. The stepwise assembly process of the immunosensor was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. Assay conditions that were optimized included the amount of chitosan nanoparticles in the preparation of carbon paste electrode, antibody concentration, and the incubation time of the antibody immobilization. Using hexacyanoferrate as a mediator, the current change increased with the concentration of human immunoglobulin G. A linear relationship in the concentration range 0.3 to 120 ng mL^?1 was achieved, with a detection limit of 0.1 ng mL^?1 (S/N?=?3). The method combines the specificity of the immunological reaction with the sensitivity of the gold colloid amplified electrochemical detection, and it has potential application in clinical immunoassay.     

Immunosensors Based on Layer‐by‐Layer Self‐Assembled Au Colloidal Electrode for the Electrochemical Detection of Antigen

Colloidal Au particles have been deposited on the gold electrode through layer‐by‐layer self‐assembly using cysteamine as cross‐linkers. Self‐assembly of colloidal Au on the gold electrode resulted in an easier attachment of antibody, larger electrode surface and ideal electrode behavior. The redox reactions of [Fe(CN)₆]^4?/[Fe(CN)₆]^3? on the gold surface were blocked due to antibody immobilization, which were investigated by cyclic voltammetry and impedance spectroscopy. The interaction of antigen with grafted antibody recognition layers was carried out by soaking the modified electrode into a phosphate buffer at pH 7.0 with various concentrations of antigen at 37 °C for 30 min. Further, an amplification strategy to use biotin conjugated antibody was introduced for improving the sensitivity of impedance measurements. Thus, the sensor based on this immobilization method exhibits a large linear dynamic range, from 5–400 μg/L for detection of Human IgG. The detection limit is about 0.5 μg/L.     

Salinomycin Derivatives Kill Breast Cancer Stem Cells by Lysosomal Iron Targeting

Salinomycin ( 1 ) exhibits a large spectrum of biological activities including the capacity to selectively eradicate cancer stem cells (CSC), making it and its derivatives promising candidates for the development of drug leads against CSC. It has been previously shown that salinomycin and its C20-propargylamine derivative (Ironomycin ( 2 )) accumulate in lysosomes and sequester iron in this organelle. Herein, a library of salinomycin derivatives is reported, including products of C20-amination, C1-esterification, C9-oxidation, and C28-dehydration. The biological activity of these compounds is evaluated against transformed human mammary epithelial HMLER CD24^low/CD44^high cells, a well-established model of breast CSC, and HMLER CD24^high/CD44^low cells deprived of CSC properties. Unlike other structural alterations, derivative 4 , which displays a cyclopropylamine at position C20, showed a strikingly low IC₅₀ value of 23 nm against HMLER CD24^low/CD44^high cells. This study provides highly selective molecules to target the CSC niche, a potential interesting advance for drug development to prevent cancer resistance.     

Development of an Amperometric Immunosensor for the Quantification of Staphylococcus aureus Using Self‐Assembled Monolayer‐Modified Electrodes as Immobilization Platforms

Amperometric immunosensors for the detection and quantification of S. aureus using MPA self‐assembled monolayer modified electrodes for the immobilization of the immunoreagents are reported. Two different immunosensor configurations were compared. A competitive mode, in which protein A‐bearing S. aureus cells and antiRbIgG labeled with horseradish peroxidase (HRP) compete for the binding sites of RbIgG immobilized onto the 3‐mercaptopropionic acid (MPA) modified electrode, was evaluated. Moreover, a sandwich configuration in which S. aureus cells were immobilized onto the MPA SAM, and RbIgG and antiRbIgG labeled with HRP were further linked to the electrode surface, was also tested. In both cases, TTF was used as the redox mediator of the HRP reaction with H₂O₂, and it was co‐immobilized onto the MPA‐modified gold electrode. After optimization of the working variables for both configurations, the analytical performance of the amperometric measurements carried out at 0.00 V (vs. Ag/AgCl) showed that the competitive immunosensor exhibited a lower limit of detection (1.6×10⁵ S. aureus cells mL^?1), as well as a better repeatability and reproducibility of the measurements.     

A Cyclometalated IrIII Complex Conjugated to a Coumarin Derivative Is a Potent Photodynamic Agent against Prostate Differentiated and Tumorigenic Cancer Stem Cells

A cyclometalated Ir^III complex conjugated to a far-red-emitting coumarin, Ir^III-COUPY ( 3 ), was recently shown as a very promising photosensitizer suitable for photodynamic therapy of cancer. Therefore, the primary goal of this work was to deepen knowledge on the mechanism of its photoactivated antitumor action so that this information could be used to propose a new class of compounds as drug candidates for curing very hardly treatable human tumors, such as androgen resistant prostatic tumors of metastatic origin. Conventional anticancer chemotherapies exhibit several disadvantages, such as limited efficiency to target cancer stem cells (CSCs), which are considered the main reason for chemotherapy resistance, relapse, and metastasis. Herein, we show, using DU145 tumor cells, taken as the model of hormone-refractory and aggressive prostate cancer cells resistant to conventional antineoplastic drugs, that the photoactivated conjugate 3 very efficiently eliminates both prostate bulk (differentiated) and prostate hardly treatable CSCs simultaneously and with a similar efficiency. Notably, the very low toxicity of Ir^III-COUPY conjugate in the prostate DU145 cells in the dark and its pronounced selectivity for tumor cells compared with noncancerous cells could result in low side effects and reduced damage of healthy cells during the photoactivated therapy by this agent. Moreover, the experiments performed with the 3D spheroids formed from DU145 CSCs showed that conjugate 3 can penetrate the inner layers of tumor spheres, which might markedly increase its therapeutic effect. Also interestingly, this conjugate induces apoptotic cell death in prostate cancer DU145 cells associated with calcium signaling flux in these cells and autophagy. To the best of our knowledge, this is the first study demonstrating that a photoactivatable metal-based compound is an efficient agent capable of killing even hardly treatable CSCs.     

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.     

Voltammetric Label‐free Immunosensors for the Diagnosis of Cystic Echinococcosis

Cystic echinococcosis (CE) or hydatid disease is a parasitic infection caused by Echinococcus granulosus. Early serodiagnosis and continuous monitoring of the disease is very important for medical treatment. Here, we report the detecting of both echinococcus antigen and antibody for the diagnosis of hydatid disease using square wave voltammetry (SWV)‐based immunosensors. The gold electrodes were functionalized using cysteamine/phenylene diisothiocyanate linkers and used for the immunosensors fabrication. The hydatid antigen and antibody immunosensors were constructed by the immobilization of either purified rabbit polyclonal antibody or recombinant antigen B (AgB), respectively on the functionalized gold electrodes surfaces. The detection in both cases was achieved by following the change in the SWV reduction peak current of the ferro/ferricyanide redox couple upon antibody or antigen binding. These immunosensors enabled the detection of echinococcus antigen and antibody within a concentration range of 1 pg.mL^?1 to 1 μg.mL^?1 with detection limits of 0.4 pg.mL^?1 and 0.3 pg.mL^?1, respectively. A preliminary application of the developed immunosensor was performed in spiked serum sample showing good recovery percentages ranging from 102 to 110 % for both hydatid antibody and antigen detection. This easy‐to‐use, sensitive, and low cost quantitative method holds great promise for the early diagnosis of hydatid disease and thus, better managements and treatment outcomes.     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

Potentiometric Immunosensor Based on Immobilization of Hepatitis B Surface Antibody on Platinum Electrode Modified Silver Colloids and Polyvinyl Butyral as Matrixes

A highly sensitive immunosensor based on immobilization of hepatitis B surface antibody (HBsAb) on platinum electrode (Pt) modified silver colloids and polyvinyl butyral (PVB) as matrixes has been developed for potentiometric immunoanalysis to detect hepatitis B surface antigen (HBsAg) in this study. HBsAb molecules were immobilized successfully on nanometer‐sized silver colloid particles associated with polyvinyl butyral on a platinum electrode surface. The modification procedure was electrochemically monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The HBsAb‐silver‐PVB‐modified electrode exhibited direct electrochemical behavior toward HBsAg. The factors influencing the performance of the resulting immunosensor were studied in detail. More than 94.7% of the results of human serum samples obtained by this method were in agreement with those obtained by enzyme‐linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited a sigmoid curve with log HBsAg concentration, high sensitivity (39.8 mV/decade), wide linear range from 16.0 to 800 ng mL^?1 with a detection limit of 3.6 ng mL^?1, fast potentiometric response (<3 min) and long‐term stability (>4 months). The response mechanism of the immunosensors was also studied with AC impedance techniques.     

Aptamer‐based Biosensor Developed to Monitor MUC1 Released by Prostate Cancer Cells

Electrochemical aptasensors can detect cancer biomarkers such as mucin 1 (MUC1) to provide point‐of‐care diagnosis that is low‐cost, specific and sensitive. Herein, a DNA hairpin containing MUC1 aptamer was thiolated, conjugated with methylene blue (MB) redox tag, and immobilized on a gold electrode by self‐assembly. The fabrication process was characterized by scanning electron microscopy, X‐ray spectroscopy analysis and electrochemistry techniques. The results evidenced a stable and sensitivity sensor presenting wide linear detection range (0.65–110 ng/mL). Therefore, it was able to precisely detect MUC1 production patterns in normal (RWPE‐1) and prostate cancer cells (LNCaP and PC3). The biosensor has ability to detect MUC1 in complex samples being an efficient and useful platform for cancer diagnosing in early stages and for physiological applications such as cancer treatment monitoring.