Gold Nanoparticle-Based Microfluidic Chips for Capture and Detection of Circulating Tumor Cells.

Liquid biopsy has progressed to its current use to diagnose and monitor cancer. Despite the recent advances in investigating cancer detection and diagnosis strategies, there is still a room for improvements in capturing CTCs. We developed an efficient CTC detection system by integrating gold nanoparticles with a microfluidic platform, which can achieve CTC capture within 120 min. Here, we report our development of a simple and effective way to isolate CTCs using antibodies attached on gold nanoparticles to the surface of a lateral filter array (LFA) microdevice. Our method was optimized using three pancreatic tumor cell lines, enabling the capture with high efficiency (90% ± 3.2%). The platform was further demonstrated for isolating CTCs from patients with metastatic pancreatic cancer. Our method and platform enables the production of functionalized, patterned surfaces that interact with tumor cells, enhancing the selective capture of CTCs for biological assays.

Recent Advances in Functional Nanomaterials for Diagnostic and Sensing Using Self-Assembled Monolayers.

Functional nanomaterials have attracted attention by producing different structures in any field. These materials have several potential applications, including medicine, electronics, and energy, which provide many unique properties. These nanostructures can be synthesized using various methods, including self-assembly, which can be used for the same applications. This unique nanomaterial is increasingly being used for biological detection due to its unique optical, electrical, and mechanical properties, which provide sensitive and specific sensors for detecting biomolecules such as DNA, RNA, and proteins. This review highlights recent advances in the field and discusses the fabrication and characterization of the corresponding materials, which can be further applied in optical, magnetic, electronic, and sensor fields.

Detection of Prostate Cancer Biomarker PCA3 by Using Aptasensors.

BACKGROUND: Prostate cancer cells have very high PCA3 messenger RNA levels, which turns them into one of the new biomarkers for prostate cancer prognosis and diagnosis. OBJECTIVE: Our goal here is to develop a new aptasensor to detect PCA3 release by the cancer cell. METHODS: DNA hairpin containing PCA3 aptamer was thiolated, conjugated to methylene blue (MB) redox probe, and immobilized on gold electrode through self-assembly to detect label-free cancer cells. RESULTS: Our data have evidenced stable and sensitive sensors presenting a wide linear detection range (0-150ng/mL). In addition, monitoring PCA3 released by different types of prostate cells can provide in-depth knowledge about prostate cancer dynamics; therefore, it is a powerful platform for earlier clinical diagnostic. The released PCA3 can vary depending on the type of adopted prostate cells. CONCLUSION: PCA3 release was monitored in a group of cells for 2 h; it showed significantly higher expression in both LNCaP and PC-3 cells. This strategy provides a unique and simple methodology to achieve more sensitive and specific PCA3 detection; thus, it emerged as a promising tool for early cost-effective diagnosis.

A global pollutant (PVC-polyvinyl chloride) applied as heavy metal binder from aqueous samples: green principles from synthesis to application.

We have developed a clean route for the modification of polyvinylchloride surface (PVC) with 4-amino-5-hydrazino-1,2,4-triazole-3-thiol molecule. The modification reaction was investigated through Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS) analysis. According to our findings, S-H groups are responsible to the molecule attachment and nitrogen atoms are directly involved in metal ion coordination. These results are in agreement with the pseudo-second-order kinetic model, which infers that chemisorption is the main mechanism for metal removal. Adsorption isotherms of Cd(II), Cu(II) and Pb(II) follow the Langmuir model and the results indicated that Ns values are 0.39, 0.52 and 0.15 mmol g-1, respectively. The calculated Ømax values for Cu(II), Pb(II) and Cd(II) were 3.93, 2.95 and 1.13, respectively, indicating that three types of complex are formed depending on the adsorbed species. Therefore, it can be concluded that PVC use as adsorbent is feasible since it requires a simple modification reaction with nontoxic and low-cost solvents.

Enzyme Immobilization on Maghemite Nanoparticles with Improved Catalytic Activity: An Electrochemical Study for Xanthine.

Generally, enzyme immobilization on nanoparticles leads to nano-conjugates presenting partially preserved, or even absent, biological properties. Notwithstanding, recent research demonstrated that the coupling to nanomaterials can improve the activity of immobilized enzymes. Herein, xanthine oxidase (XO) was immobilized by self-assembly on peculiar naked iron oxide nanoparticles (surface active maghemite nanoparticles, SAMNs). The catalytic activity of the nanostructured conjugate (SAMN@XO) was assessed by optical spectroscopy and compared to the parent enzyme. SAMN@XO revealed improved catalytic features with respect to the parent enzyme and was applied for the electrochemical studies of xanthine. The present example supports the nascent knowledge concerning protein conjugation to nanoparticle as a means for the modulation of biological activity.

A Methodology for Porcine Circovirus 2 (PCV-2) Quantification Based on Gold Nanoparticles.

The aim of the current study is to introduce a methodology aimed at producing a biosensor that uses gold nanoparticles (AuNPs) to detect porcine circovirus 2 (PCV-2). This biosensor was based on AuNPs, which were modified with self-assembled monolayers (SAMs) and antibodies. The AuNPs' surface and virus modification process applied to enable antibody binding was accompanied by localized surface plasmon resonance (LSPR), surface plasmon resonance (SPR), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDX). Virus quantification was possible by the light absorption difference in the spectrum at concentrations of 105, 106, 107, 108, and 109 DNA copies/mL PCV-2 in relation to quantitative PCR (qPCR), with an R2 value >0.98. The visualization of colorimetric changes in the different PCV-2 concentrations was possible without the use of equipment. The biosensor production methodology presented reproducibility and specificity, as well as easy synthesis and low cost. An enhanced version of it may be used in the future to replace traditional tests such as PCR.

Anti-EGFR-Coated Gold Nanoparticles In Vitro Carry 5-Fluorouracil to Colorectal Cancer Cells.

: The aim of the current study is to present a strategy to improve the efficiency of 5-fluorouracil (5-FU), which is widely used as antineoplastic agent against solid tumors-based on the use of gold nanocarriers to overcome the resistance of colorectal cancer cells. 5-FU was loaded on gold nanoparticles (AuNP) coated with anti-EGFR antibodies in order to target them towards colorectal cancer cells that overexpress epidermal growth factor receptors (EGFR). Physicochemical characterization has shown that AuNP size was approximately 20 nm and that AuNP functionalization led to spherical nanoparticles. Flow cytometry allowed observing that some compounds synthesized by our research group have induced apoptosis/necrosis and impaired the proliferation of colon cancer cell lines 'HCT-116' and 'HT-29'. The antibody/drug combination in AuNP (AuNP 5FU EGFR) has improved the apoptosis rate and impaired cell proliferation in both cell lines, regardless of the exposure time. Overall, these results have shown that AuNP functionalization with monoclonal antibodies focused on delivering 5-FU to tumor cells is an exciting strategy against colorectal cancer.

Electrocatalytic nanostructured ferric tannate as platform for enzyme conjugation: Electrochemical determination of phenolic compounds.

A shell of nanostructured ferric tannates was spontaneously developed on the surface of naked maghemite nanoparticles (SAMNs, the core) by a simple wet reaction with tannic acid (TA). The as obtained core-shell nanomaterial (SAMN@TA) displays specific electrocatalytic and surface properties, which significantly differ from parent maghemite. Thanks to the known proclivity of TA to interact with proteins, SAMN@TA was proposed as a support for the direct immobilization of an enzyme. A ternary functional nanobioconjugate (SAMN@TA@TvL) was successfully self-assembled by incubating laccase from Trametes versicolor (TvL) and SAMN@TA. The SAMN@TA@TvL hybrid was kinetically characterized with respect to the native enzyme and applied for building an easy-to-use analytical device for the detection of polyphenols. The electrochemical biosensor allowed the determination of polyphenols by square wave voltammetry in mixed water-methanol solutions. The system sensitivity was 868.9 ±â€¯1.9nA µM-1, the LOD was 81 nM and the linearity range was comprised between 100 nM and 10 µM. The proposed approach was successfully applied to detect phenolics in blueberry extracts as real samples. Results suggest that SAMN@TA could be a promising, low cost and versatile tool for the creation of nano-bio-conjugates aimed at the development of new electrochemical sensing platforms.

A new immunoassay of hybrid nanomater conjugated to aptamers for the detection of dengue virus.

A new immunosensor using hybrid nanomaterials for the detection of dengue virus was demonstrated in this work. This immunosensor composed of nanoparticles of γ-Fe2O3(SAMN) modified with MPA- SAMN@MPA was characterized by FTIR spectroscopy, transmission electron microscopy,quartz crystal microbalance, UV-vis and LSPR technique. The binding of SAMN@MPA with AuNPs conjugated with aptamers(SAMN@MPA@AuNPs@aptamer) provides specific chemical bonds to four dengue serotypes. Colorimetric changes in the modification steps provided rapid visual detection of the virus without the use of equipment. Variations of aptamers concentrations 1.0-10.0 µM where the 3.0 µM aptamer concentration is sufficient to completely cover the surface of the modified AuNPs with an R2 value of> 0.99. This new proposed methodology presenting some advantages in relation to traditional detection methods such as time optimization and cost,can be used as a diagnostic method.

An easy way to detect dengue virus using nanoparticle-antibody conjugates.

The aim of the present research is to propose a new method based on localized surface plasmon resonance (LSPR) for fast dengue virus detection. A pool with four dengue serotypes (DENV-1, -2, -3, -4) was detected through antigen-antibody binding using gold nanoparticles (AuNPs) as signaling antibody carriers. Such result was confirmed through surface plasmon resonance (SPR), transmission electron microcopy (TEM), and dynamic light scattering (DLS) techniques. The limit of detection was calculated for TCID50 107 demonstrating a linear correlation between viral concentration and number of cells with an r2 value of > 0.993. The assay presented good sensibility and reproducibility of results and the negative controls were not mistakenly detected. This design requires no pretreatment or high trained person. In the future, it can be used in commercial antibody detection kits.

Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells.

Early prostate cancer (PCa) diagnostic is crucial to enhance patient survival rates; besides, non-invasive platforms have been developed worldwide in order to precisely detect PCa biomarkers. Therefore, the aim of the present study is to develop a new aptamer-based biosensor through the self-assembling of thiolated aptamers for PSA and VEGF on the top of gold electrodes. This biosensor was tested in three prostate cell lines (RWPE-1, LNCaP and PC3). The results evidenced a stable and sensitive sensor presenting wide linear detection ranges (0.08-100 ng/mL for PSA and 0.15 ng-100 ng/mL for VEGF). Therefore, the aptasensor was able to detect the patterns of PSA and VEGF released in vitro by PCa cells, which gave new insights about the prostate cancer protein dynamics. Thus, it could be used as a non-invasive PCa clinical diagnosis instrument in the near future. Graphical Abstract Overview of the experimental design applied to the aptamer-based electrochemical sensor self-assembled on the thiolated hairpin structure. A filter membrane was added on top of working electrode to provide the cell-attachment surface after aptamer incubation, without compromising the aptamer layer. The pore membrane allowed target proteins to pass to the aptamer surface; the MCH backfilling avoided unspecific protein binding to the gold electrode surface.

Development of a rapid and sensitive immunosensor for the detection of bacteria.

The presence of Gram-positive bacteria in foodstuffs is a chronic worldwide problem. Here, we present a cheap and simple colorimetric method for the detection of Lactobacillus species (spp.) and Staphylococcus aureus (S. aureus) using gold nanoparticles (AuNP) modified with monoclonal anti-Gram-positive bacteria to produce an immune-sensor. Detection is based on the fact that antibody-conjugated AuNPs can readily identify Gram-positive bacteria through antibody-antigen recognition, which results in a color change of AuNPs upon aggregation. The detection limit was 105CFU/ml in pure culture for Lactobacillus spp. and 120CFU/ml in pure culture for S. aureus. The method was applied successfully for detection of bacteria in samples of sugar cane, and agreed well with values obtained using other methods. These results suggested that the detection system could be used for the quantitative analysis of Gram-positive bacteria and might be applied potentially by the food industry.

Glucose biosensor based on multisegment nanowires exhibiting reversible magnetic control.

We describe the amperometric detection of glucose using oriented nanowires with magnetic switching of the bioelectrochemical process. The fabrication process of the nanowires was prepared through controlled nucleation and growth during a stepwise electrochemical deposition, and it was characterized using scanning electron microscopy. Cyclic voltammetry and amperometry were used to study the magnetoswitchable property; this control was accomplished by changing the surface orientation of nanowires. Under the optimal condition, the amperometric response was also linear up to a glucose concentration of 0.1-16.0 mmol L(-1) with a sensitivity of 81 µA mM(-1). The detection limit was estimated for 4.8×10(-8) mol L(-1), defined from a signal/noise ratio of 3. It also exhibits good reproducibility and high selectivity with insignificant interference from ascorbic acid, acetoaminophen, and uric acid. The resulting biosensor was applied to detect the blood sugar in human serum samples without any pretreatment, and the results were comparatively in agreement with the clinical assay.

Plasmonic nanobiosensor with chain reaction amplification mechanism.

Herein we demonstrate a plasmonic nanobiosensor that explores chain reaction amplification mechanisms to transduce chemical signals released in biocatalytic reactions, turning optical signals into a visual spectral range. The sensor has a very simple design: gold nanoparticles resting in the surface of a grafted P2VP film. Changes in the gold nanoparticles' position causes changes in the plasmon coupling mode. This is detected by means of a maximum absorbance shift.

Electrochemical biosensors for on-chip detection of oxidative stress from immune cells.

Seamless integration of biological components with electrochemical sensors is critical in the development of microdevices for cell analysis. The present paper describes the integration miniature Au electrodes next to immune cells (macrophages) in order to detect cell-secreted hydrogen peroxide (H(2)O(2)). Photopatterning of poly(ethylene glycol) (PEG) hydrogels was used to both immobilize horseradish peroxidase molecules onto electrodes and to define regions for cell attachment in the vicinity of sensing electrodes. Electrodes micropatterned in such a manner were enclosed inside poly(dimethylsiloxane) fluid conduits and incubated with macrophages. The cells attached onto the exposed glass regions in the vicinity of the electrodes and nowhere else on the non-fouling PEG hydrogel surface. A microfluidic device was converted into an electrochemical cell by placing flow-through Ag∕AgCl reference and Pt wire counter electrodes at the outlet and inlet, respectively. This microdevice with integrated H(2)O(2)-sensing electrodes had sensitivity of 27 µA∕cm(2) mM with a limit of detection of 2 µM. Importantly, this microdevice allowed controllable seeding of macrophages next to electrodes, activation of these cells and on-chip monitoring of H(2)O(2) release in real time. In the future, this biosensor platform may be utilized for monitoring of macrophage responses to pathogens or for the study of inflammatory signaling in micropatterned cell cultures.

Spectrofluorimetric determination of coumarin in commercial tablets.

A simple, rapid and effective analytical method based on fluorescence spectroscopy for the determination of coumarin in pharmaceutical formulations without pre-treatment or pre-concentration step was development. Coumarin had maximum excitation and emission at 310 nm and 390 nm, respectively. Optimum conditions for the detection of coumarin were investigated. Under optimized conditions, we observed a linear behavior for the sign of coumarin in the concentration range of 2.5 × 10(-6) to 1.0 × 10(-4) mol L(-1), with linearity of 0.998 and sensitivity of 2.9 × 10(10) u.a/mol L(-1). The proposed method was validated in terms of accuracy, precision and specificity of coumarin using the standard addition and external calibration. It was noted that the results support (P<0.05), indicating that the matrices were not an interference in the determination of coumarin by fluorescence spectroscopy. The results were favorable compared with those obtained by reference chromatographic methods.

Renewable nanocomposite layer-by-layer assembled catalytic interfaces for biosensing applications.

A novel, easily renewable nanocomposite interface based on layer-by-layer (LbL) assembled cationic/anionic layers of carbon nanotubes customized with biopolymers is reported. A simple approach is proposed to fabricate a nanoscale structure composed of alternating layers of oxidized multiwalled carbon nanotubes upon which is immobilized either the cationic enzyme organophosphorus hydrolase (OPH; MWNT-OPH) or the anionic DNA (MWNT-DNA). The presence of carbon nanotubes with large surface area, high aspect ratio and excellent conductivity provides reliable immobilization of enzyme at the interface and promotes better electron transfer rates. The oxidized MWNTs were characterized by thermogravimetric analysis and Raman spectroscopy. Fourier transform infrared spectroscopy showed the surface functionalization of the MWNTs and successful immobilization of OPH on the MWNTs. Scanning electron microscopy images revealed that MWNTs were shortened during sonication and that LbL of the MWNT/biopolymer conjugates resulted in a continuous surface with a layered structure. The catalytic activity of the biopolymer layers was characterized using absorption spectroscopy and electrochemical analysis. Experimental results show that this approach yields an easily fabricated catalytic multilayer with well-defined structures and properties for biosensing applications whose interface can be reactivated via a simple procedure. In addition, this approach results in a biosensor with excellent sensitivity, a reliable calibration profile, and stable electrochemical response.

Immobilizing enzymes onto electrode arrays by hydrogel photolithography to fabricate multi-analyte electrochemical biosensors.

This paper describes a biomaterial microfabrication approach for interfacing functional biomolecules (enzymes) with electrode arrays. Poly (ethylene glycol) (PEG) hydrogel photopatterning was employed to integrate gold electrode arrays with the enzymes glucose oxidase (GOX) and lactate oxidase (LOX). In this process, PEG diacrylate (DA)-based prepolymer containing enzyme molecules as well as redox species (vinylferrocene) was spin-coated, registered, and UV cross-linked on top of an array of gold electrodes. As a result, enzyme-carrying circular hydrogel structures (600 microm diameter) were fabricated on top of 300 microm diameter gold electrodes. Importantly, when used with multiple masks, hydrogel photolithography allowed us to immobilize GOX and LOX molecules on adjacent electrodes within the same electrode array. Cyclic voltammetry and amperometry were used to characterize biosensor electrode arrays. The response of the biosensor array was linear for up to 20 mM glucose with sensitivity of 0.9 microA cm(-2) mM(-1) and 10 mM lactate with sensitivity of 1.1 microA cm(-2) mM(-1). Importantly, simultaneous detection of glucose and lactate from the same electrode array was demonstrated. A novel strategy for integrating biological and electrical components of a biosensor described in this paper provides the flexibility to spatially resolve and register different biorecognition elements with individual members of a miniature electrode array. Of particular interest to us are future applications of these miniature electrodes for real-time monitoring of metabolite fluxes in the vicinity of living cells.

Enhanced stability of enzyme organophosphate hydrolase interfaced on the carbon nanotubes.

In this paper we demonstrate that SWNTs and a covalent immobilization strategy enable very sensitive sensors with excellent long term stability. Organophosphorus hydrolase (OPH) functionalized single and multi-walled carbon nanotube (CNT) conjugates were exploited for direct amperometric detection of paraoxon, a model organophosphate. The catalytic hydrolysis of paraoxon produces equimoles of p-nitrophenol; oxidation was monitored amperometrically in real time under flow-injection (FIA) mode. OPH covalently immobilized on single-walled carbon nanotubes (SWNTs) demonstrated much higher activity than OPH conjugated to multi-walled carbon nanotubes (MWNTs). The dynamic concentration range for SWNT-OPH was 0.5-8.5 micromolL(-1) with a detection limit of 0.01 micromolL(-1) (S/N=3). In addition to this high sensitivity, the immobilized OPH retained a significant degree of enzymatic activity, and displayed remarkable stability with only 25% signal loss over 7 months. These results suggest that covalent immobilization of OPH on CNTs can be used for specific immobilization with advantages of long term stability, high sensitivity, and simplicity.

Enzymatic nanolithography of polyaniline nanopatterns by using peroxidase-modified atomic force microscopy tips.

A new enzyme nanolithography strategy for creating conducting polymer nanostructures through the modification of AFM tips with peroxidase is described. Scanning of the modified tip in the presence of aniline and hydrogen peroxide is used for biocatalytic patterning of different polyaniline nanostructures (see figure).