Microfluidic-based blood immunoassays.

Microfluidics enables the integration of whole protocols performed in a laboratory, including sample loading, reaction, extraction, and measurement steps on a single system, which offers significant advantages thanks to small-scale operation combined with precise fluid control. These include providing efficient transportation mechanisms and immobilization, reduced sample and reagent volumes, fast analysis and response times, lower power requirements, lower cost and disposability, improved portability and sensitivity, and greater integration and automation capability. Immunoassay is a specific bioanalytical method based on the interaction of antigens and antibodies, which is utilized to detect bacteria, viruses, proteins, and small molecules in several areas such as biopharmaceutical analysis, environmental analysis, food safety, and clinical diagnostics. Because of the advantages of both techniques, the combination of immunoassays and microfluidic technology is considered one of the most potential biosensor systems for blood samples. This review presents the current progress and important developments in microfluidic-based blood immunoassays. After providing several basic information about blood analysis, immunoassays, and microfluidics, the review points out in-depth information about microfluidic platforms, detection techniques, and commercial microfluidic blood immunoassay platforms. In conclusion, some thoughts and future perspectives are provided.

An ECL sensor combined with a paper electrode for the determination of phenylalanine.

Herein, a unique electrochemiluminescence (ECL) sensor combined with a paper electrode was proposed for the detection of phenylalanine (L-Phe) in blood samples. The L-Phe detection was performed by converting L-Phe into ammonia using phenylalanine ammonia-lyase (PAL) enzyme and the ECL signal of Ru(bpy)32+ was produced in combination with ammonia as a co-reactant. In this report, we used AuNP decorated paper electrodes to obtain the ECL signal of Ru-(bpy)32+ in the presence of NH3. The produced ammonia molecules were carried to the working electrode and their contact with the sample was cut off. Since there was no contact between the sample matrix and the electrode, the developed method achieved excellent selectivity. According to experimental data, a linear increase of the ECL signals with the logarithms of varying L-Phe concentrations between 1.5 × 10-2 and 1.211 mM was observed with a correlation coefficient (R2) of 0.9898 and a limit of detection (LOD) of 8.4 × 10-3 mM. The proposed method was efficiently applied for L-Phe detection in reference blood samples and the average recovery was calculated as 96.8%. Furthermore, the HPLC-UV method as a comparison verified that the recovery values provided by the proposed method were acceptable with a similarity of 95.1% for L-Phe detection in a reference blood sample. The results showed that the developed ECL sensor combined with the paper electrode can be considered as a promising unique and powerful technique for a point-of-care diagnosis of PKU.

Impedimetric detection of miRNA biomarkers using paper-based electrodes modified with bulk crystals or nanosheets of molybdenum disulfide.

Paper-based electrodes modified with molybdenum disulfide (MoS2) in the form of bulk crystals or exfoliated nanosheets were developed and used as a biosensing platform for the impedimetric detection of miRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. For this purpose, MoS2 crystals or nanosheets were used for the modification of the working electrode area of paper-based platform for the first time in this study. The proposed assay offers sensitive and selective detection of microRNAs by electrochemical impedance spectroscopy (EIS) technique. The entire assay, both the electrode modification and the miRNA detection being completed in 30 min and a single sample droplet (5 µL) was enough to cover working electrode area which enabled analysis in low sample volumes. The limits of detection (LOD) for miRNA-21 and miRNA-155 were calculated both in buffer and fetal bovine serum media. It is found that the LOD is varying between 1 and 200 ng/mL. In comparison to nanosheets, a larger electroactive surface area was obtained with bulk MoS2 resulting in lower LOD values on miRNA detection. The paper-based electrodes showed high specificity towards their target sequences. Moreover, they effectively discriminated single base mismatched non-target sequences. The advantages of these MoS2 paper based electrodes include high sensitivity, and low-cost provide great potential for improved monitoring of miRNA biomarkers even in artificial serum media.

A capillary driven microfluidic chip for SERS based hCG detection.

In this study, a capillary driven microfluidic chip-based immunoassay was developed for the determination of Human Chorionic Gonadotropin (hCG) protein, which is prohibited by the World Anti-Doping Agency (WADA). Here, we used antibody modified magnetic metal organic framework nanoparticles (MMOFs) as a capture prob in urine sample. MMOF captured hCG was transferred in a capillary driven microfluidic chip consisting of four chambers, and the interaction of MMOF with gold nanorods labelled with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) as a Raman label was carried out in the capillary driven microfluidic chip. The movement of MMOF through first chamber to the last chamber was achieved with a simple magnet. In the last chamber of capillary driven microfluidic chip, SERS signals of DTNB molecules from the sandwich complex were recorded using a Raman spectrophotometer. The selectivity of the developed method was demonstrated by applying the same procedure for the detection of Human Luteinizing Hormone (hLH), Human Chorionic Gonadotropin Hormone (hGH) and Immunoglobulin G (IgG) protein. The regression coefficient and limit of detection obtained from the standard addition method were found as 0,9985 and 0,61 IU/L, respectively. Furthermore, the conventional ELISA method confirmed that the results obtained by the presented method were acceptable with the similarity of 97.9% in terms of average recovery value, for the detection of hCG in urine samples. The analysis system developed for target proteins will be an alternative technique such as Western Blot used in routine analysis that is expensive and time consuming.

Paper-Based Electrochemical Biosensors for Voltammetric Detection of miRNA Biomarkers Using Reduced Graphene Oxide or MoS2 Nanosheets Decorated with Gold Nanoparticle Electrodes.

Paper-based biosensors are considered simple and cost-efficient sensing platforms for analytical tests and diagnostics. Here, a paper-based electrochemical biosensor was developed for the rapid and sensitive detection of microRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. Hydrophobic barriers to creating electrode areas were manufactured by wax printing, whereas a three-electrode system was fabricated by a simple stencil approach. A carbon-based working electrode was modified using either reduced graphene oxide or molybdenum disulfide nanosheets modified with gold nanoparticle (AuNPs/RGO, AuNPs/MoS2) hybrid structures. The resulting paper-based biosensors offered sensitive detection of miRNA-155 and miRNA-21 by differential pulse voltammetry (DPV) in only 5.0 µL sample. The duration in our assay from the point of electrode modification to the final detection of miRNA was completed within only 35 min. The detection limits for miRNA-21 and miRNA-155 were found to be 12.0 and 25.7 nM for AuNPs/RGO and 51.6 and 59.6 nM for AuNPs/MoS2 sensors in the case of perfectly matched probe-target hybrids. These biosensors were found to be selective enough to distinguish the target miRNA in the presence of single-base mismatch miRNA or noncomplementary miRNA sequences.

Paper-based electrode assemble for impedimetric detection of miRNA.

In the present work, a paper-based electrode assemble was developed and implemented to detect target microRNA 155 (miRNA 155) via electrochemical impedance spectroscopy (EIS) measurements. In this concept, gold nanoparticles (AuNPs) modified paper based electrode assemble system (AuNP-PE) was designed, and characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and EIS measurements. The impedimetric detection of miRNA 155 was performed by measuring the fractional change at the charge transfer resistance (Rct). The detection limits were found as 33.8 nM in PBS and 93.4 nM in fetal bovine serum (FBS) medium, respectively. The selectivity of the proposed assay was tested against to non-complementary (NC) and mismatch (MM) miRNA sequences in the presence of mixture sample containing miRNA:NC (1:1) and miRNA:MM (1:1) in PBS (pH 7.40) or FBS. The analytical performance and the selectivity of impedimetric biosensor were also tested in FBS.

Immunomagnetic separation and Listeriamonocytogenes detection with surface-enhanced Raman scattering

Background/aim: We aimed to develop a rapid method to enumerate Listeria monocytogenes (L. monocytogenes) utilizing magnetic nanoparticle based preconcentration and surface-enhanced Raman spectroscopy measurements. Materials and methods: Biological activities of magnetic Au-nanoparticles have been observed to have the high biocompatibility, and a sample immunosensor model has been designed to use avidin attached Au-nanoparticles for L. monocytogenes detection. Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhimurium) bacteria cultures were chosen for control studies. Antimicrobial activity studies have been done to identify bio-compatibility and bio-characterization of the Au-nanoparticles in our previous study and capturing efficiencies to bacterial surfaces have been also investigated. Results: We constructed the calibration graphs in various population density of L. monocytogenes as 2.2 × 101 to 2.2 × 106 cfu/mL and the capture efficiency was found to be 75%. After the optimization procedures, population density of L. monocytogenes and Raman signal intensity showed a good linear correlation (R2 = 0.991) between 102 to 106 cfu/mL L. monocytogenes. The presented sandwich assay provides low detection limits and limit of quantification as 12 cfu/mL and 37 cfu/mL, respectively. We also compared the experimental results with reference plate-counting methods and the practical utility of the proposed assay is demonstrated using milk samples. Conclusion: It is focused on the enumeration of L. monocytogenes in milk samples and the comparision of results of milk analysis obtained by the proposed SERS method and by plate counting method stay in food agreement. In the present study, all parameters were optimized to select SERS-based immunoassay method for L. monocytogenes bacteria to ensure LOD, selectivity, precision and repeatablity.

The coupling of immunomagnetic enrichment of bacteria with paper-based platform.

In this study, the coupling of magnetic enrichment of bacteria from real samples with rapid surface enhanced Raman spectroscopy (SERS) detection was reported. The selective isolation and enrichment for the model bacteria Escherichia coli (E. coli) was performed using E. coli (primary) antibody bound-magnetic gold (Fe3O4@Au) nanoparticles. Following isolation and enrichment, the rennet enzyme was used to cleave of casein modified Fe3O4/Au-PEI nanoparticles from primary antibody-bound bacteria to prevent the nanoparticle aggregation and provide the movement of bacteria on nitrocellulose membrane. In the first part of the study, optimization studies were carried out namely; the amounts of gold nanoparticles (AuNPs), polyethyleneimine coated magnetic gold (Fe3O4/Au-PEI) nanoparticles, casein and rennet enzyme. The SERS signals of DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)) molecule were collected on the test line and a calibration curve was plotted by using signal intensities. The correlation between the concentration of E. coli and SERS signal was found to be linear within the range of 101-107 cfu/mL (R2 = 0.984, LOD = 0.52 cfu/mL and LOQ = 1.57 cfu/mL). The selectivity of the paper-based lateral flow immunoassay (LFIA) was examined with Bacillus subtilis (B. subtilis), Micrococcus luteus (M. luteus), Salmonella enteritidis (S. enteritidis) which did not produce any significant response compared with E. coli measurement. Finally, the developed paper-based LFIA was tested with urine and milk samples. The obtained SERS results were compared with a plate counting method results which were in a good accordance. The developed method was found as rapid and sensitive to E. coli with a total analysis time of less than 60 min.

Investigation of eluted monomers from resin-based root canal sealer by high-performance liquid chromatography analysis.

OBJECTIVE: The purpose of the current study was to determine the amount of urethane dimethacrylate (UDMA), bisphenol A-glycidyl methacrylate (Bis-GMA), poly (ethylene glycol) dimethacrylate (PEGDMA), bisphenol A ethoxylated dimethacrylate (Bis-EMA), and 2-hydroxyethyl methacrylate (HEMA) eluted from resin-based root canal sealer, epiphany, using high-performance liquid chromatography (HPLC). MATERIALS AND METHODS: Epiphany was placed into the plastic molds and light-cured with a light emitting diode. After the curing process, each specimen in the first group (n = 12) was immersed in Eppendorf tubes containing a phosphate-buffered saline solution (PBS) and incubated for 45 s. In the second group, each specimen (n = 12) was immersed in Eppendorf tubes containing PBS and incubated for 24 h. Of the specimen extracts, 100 µL were subjected to HPLC. Analysis of data was accomplished with one-way analysis of variance (P < 0.05). RESULTS: All of the samples eluted HEMA, UDMA, Bis-GMA, PEGDMA, and Bis-EMA. A significant difference was determined between the time periods of HEMA, UDMA, PEGDMA, and Bis-EMA (P < 0.05). CONCLUSION: The results of the current study showed that Epiphany releases HEMA, UDMA, Bis-GMA, PEGDMA, and Bis-EMA in both time periods.

Paper membrane-based SERS platform for the determination of glucose in blood samples.

In this report, we present a paper membrane-based surface-enhanced Raman scattering (SERS) platform for the determination of blood glucose level using a nitrocellulose membrane as substrate paper, and the microfluidic channel was simply constructed by wax-printing method. The rod-shaped gold nanorod particles were modified with 4-mercaptophenylboronic acid (4-MBA) and 1-decanethiol (1-DT) molecules and used as embedded SERS probe for paper-based microfluidics. The SERS measurement area was simply constructed by dropping gold nanoparticles on nitrocellulose membrane, and the blood sample was dropped on the membrane hydrophilic channel. While the blood cells and proteins were held on nitrocellulose membrane, glucose molecules were moved through the channel toward the SERS measurement area. Scanning electron microscopy (SEM) was used to confirm the effective separation of blood matrix, and total analysis is completed in 5 min. In SERS measurements, the intensity of the band at 1070 cm(-1) which is attributed to B-OH vibration decreased depending on the rise in glucose concentration in the blood sample. The glucose concentration was found to be 5.43 ± 0.51 mM in the reference blood sample by using a calibration equation, and the certified value for glucose was 6.17 ± 0.11 mM. The recovery of the glucose in the reference blood sample was about 88 %. According to these results, the developed paper-based microfluidic SERS platform has been found to be suitable for use for the detection of glucose in blood samples without any pretreatment procedure. We believe that paper-based microfluidic systems may provide a wide field of usage for paper-based applications.

Monosodium glutamate in chicken and beef stock cubes using high-performance liquid chromatography.

In this survey monosodium glutamate (MSG) levels in chicken and beef stock cube samples were determined. A total number of 122 stock cube samples (from brands A, B, C, D) were collected from local markets in Ankara, Turkey. High-performance liquid chromatography with diode array detection (HPLC-DAD) was used for quantitative MSG determination. Mean MSG levels (±SE) in samples of A, B, C and D brands were 14.6 ± 0.2 g kg⁻¹, 11.9 ± 0.3 g kg⁻¹, 9.7 ± 0.1 g kg⁻¹ and 7.2 ± 0.1 g kg⁻¹, respectively. Differences between mean levels of brands were significant. Also, mean levels of chicken stock cube samples were lower than in beef stock cubes. Maximum limits for MSG in stock cubes are not specified in the Turkish Food Codex (TFC). Generally the limit for MSG in foods (except some foods) is established as 10 g kg⁻¹ (individually or in combination).

A novel glucose biosensor platform based on Ag@AuNPs modified graphene oxide nanocomposite and SERS application.

This study represents a novel template demonstration of a glucose biosensor based on mercaptophenyl boronic acid (MBA) terminated Ag@AuNPs/graphene oxide (Ag@AuNPs-GO) nanomaterials. The nanocomposites were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) method. The TEM image shows that Ag@AuNPs in the nanocomposite is in the range of diameters of 10-20 nm. The nanocomposite was used for the determination of glucose through the complexation between boronic acid and diol groups of glucose. Thus, a novel glucose biosensor was further fabricated by immobilizing glucose oxidase (GOD) into MBA terminated Ag@AuNPs-GO nanocomposite film (MBA-Ag@AuNPs-GO). The linearity range of glucose was obtained as 2-6mM with detection limit of 0.33 mM. The developed biosensor was also applied successfully for the determination of glucose in blood samples. The concentration value of glucose in blood samples was calculated to be 1.97±0.002 mM from measurements repeated for six times.

Attomole sensitivity of staphylococcal enterotoxin B detection using an aptamer-modified surface-enhanced Raman scattering probe.

In this report, we present a new homogeneous detection method for staphylococcal enterotoxin B (SEB) utilizing core-shell-structured iron-gold magnetic nanoparticles and a gold nanorod surface-enhanced Raman scattering (SERS) probe in solution. Peptide ligand (aptamer) functionalized magnetic gold nanorod particles were used as scavengers for target SEB. After the SEB molecules were separated from the matrix, the sandwich assay procedure was tested by gold nanorod particles that act as SERS probes. The binding constant between SEB and peptide-nanoparticle complex was determined as 8.0 × 10(7) M(-1). The correlation between the SEB concentration and SERS signal was found to be linear within the range of 2.5 fM to 3.2 nM. The limit of detection for the homogeneous assay was determined as 224 aM (ca. 2697 SEB molecules/20 µL sample volume). Also, gold-coated surfaces were used as capture substrates and performances of the two methods were compared. Furthermore, the developed method was evaluated for investigating the SEB specificity on bovine serum albumin (BSA) and avidin and detecting SEB in artificially contaminated milk, blood, and urine.

Determination of enantiomers of atenolol and propranolol in pharmaceutical formulations by HPLC.

A validated HPLC-UV method was developed for the determination of R(-), S(+)-atenolol and R(-), S(+)-propranolol in pharmaceutical formulations. The proposed method required no elaborate sample preparation and was found to be selective, linear, and repeatable within the established ranges. Atenolol and propranolol isomers were separated using a Chirex 3022 (S) column with the mobile phases hexane-dichloromethane-methanol-trifluoroacetic acid (35 + 35 + 5 + 0.25, v/v/v/v) and hexane-dichloromethane-ethanol-trifluoroacetic acid (55 + 40 + 5 + 0.25, v/v/v/v), respectively. The LOD values of R(-) and S(+)-atenolol were 12.3 and 9.86 microg/mL, respectively, and 0.61 and 0.89 microg/mL, respectively, for R(-) and S(+)-propranolol. Retention times of R(-)-propranolol and S(+)-propranolol were 12.4 and 14.3 min, respectively, and 29.06 and 32.71 min, respectively, for (R)-atenolol and (S)-atenolol. The proposed method was applied to the determination of enantiomers in pharmaceutical formulations, and no interference from any excipients was found.