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The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody–aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated. info:eu-repo/semantics/publishedVersion

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

Drug residues in water represent one of the most serious environmental problems because of their high toxicity to the biological system. Thus, this study reports a low-cost strategy for fast and pr...

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.

The evolution in foodstuff-monitoring processes has increased the number of studies on biogenic amines (BAs), in recent years. This trend with future perspective needs to be assembled to address the associated health risks. Thus, this study aims to cover three main aspects of BAs: (i) occurrence, physiology, and toxicological effects, most probable formation mechanisms and factors controlling their growth; (ii) recent advances, strategies for determination, preconcentration steps, model technique, and nature of the matrix; and (iii) milestone, limitations with existing methodologies, future trends, and detailed expected developments for clinical use and on-site ultra-trace determination. The core of the ongoing review will discuss recent trends in pre-concentration toward miniaturization, automation, and possible coupling with electrochemical techniques, surface-enhanced Raman scattering, spectrofluorimetry, and lateral flow protocols to be exploited for the development of rapid, facile, and sensitive on-site determination strategies for BAs.

In this work, a duplex PCR–Enzyme Linked Oligonucleotide Assay (ELONA) is reported for the sensitive and reliable detection of pork adulteration in beef and chicken products, two of the most widely consumed meat types in the world. The strategy relies on the use of species-specific tailed primers for duplex amplification and simple dilution of the PCR reactions for direct colorimetric detection via hybridization, eliminating the need for any other post-amplification steps. A high sensitivity was achieved, with as low as 71–188 pg of genomic DNA able to be detected using mixtures of control DNA from each species. The strategy was validated using DNA add-mixtures as well as DNA extracted from raw meat mixtures and 0.5–1% w/w pork could be easily detected when mixed with beef or chicken. The proposed approach is simple, sensitive and cost-effective compared to equivalent commercial kits suitable for detecting adulterant pork levels in meat products.

The blood-brain barrier (BBB) is a main obstacle for drug delivery targeting the central nervous system (CNS) and treating Alzheimer's disease (AD). In order to enhance the efficiency of drug delivery without harming the BBB integrity, nanoparticle-mediated drug delivery has become a popular therapeutic strategy. Carbon dots (CDs) are one of the most promising and novel nanocarriers. In this study, amphiphilic yellow-emissive CDs (Y-CDs) were synthesized with an ultrasonication-mediated methodology using citric acid and o-phenylenediamine with a size of 3 nm that emit an excitation-independent yellow photoluminescence (PL). The content of primary amine and carboxyl groups on CDs was measured as 6.12 × 10-5 and 8.13 × 10-3 mmol mg-1, respectively, indicating the potential for small-molecule drug loading through bioconjugation. Confocal image analyses revealed that Y-CDs crossed the BBB of 5-day old wild-type zebrafish, most probably by passive diffusion due to the amphiphilicity of Y-CDs. And the amphiphilicity and BBB penetration ability didn't change when Y-CDs were coated with different hydrophilic molecules. Furthermore, Y-CDs were observed to enter cells to inhibit the overexpression of human amyloid precursor protein (APP) and β-amyloid (Aβ) which is a major factor responsible for AD pathology. Therefore, data suggest that Y-CDs have a great potential as nontoxic nanocarriers for drug delivery towards the CNS as well as a promising inhibiting agent of Aβ-related pathology of the AD.

Mycotoxins are toxic compounds produced by fungi, which represent a risk to the food and feed supply chain, having an impact on health and economies. A high percentage of feed samples have been reported to be contaminated with more than one type of mycotoxin. Systematic, cost-effective and simple tools for testing are critical to achieve a rapid and accurate screening of food and feed quality. In this review, we describe the various aptamers that have been selected against mycotoxins and their incorporation into optical and electrochemical aptasensors, outlining the strategies exploited, highlighting the advantages and disadvantages of each approach. The review also discusses the different materials used and the immobilization methods employed, with the aim of achieving the highest sensitivity and selectivity.

Pediatric glioblastomas are known to be one of the most dangerous and life-threatening cancers among many others regardless of the low number of cases reported. The major obstacles in the treatment of these tumors can be identified as the lack of prognosis data and the therapeutic requirement to be able to cross the blood-brain barrier (BBB). Due to this lack of data and techniques, pediatric patients could face drastic side effects over a long-time span even after survival. Therefore, in this study, the capability of non-toxic carbon nitride dots (CNDs) to selectively target pediatric glioblastoma cells was studied in vitro. Furthermore, the nanocarrier capability and efficiency of CNDs were also investigated through conjugation of a chemotherapeutic agent and transferrin (Tf) protein. Gemcitabine (GM) was introduced into the system as a chemotherapeutic agent, which has never been successfully used for the treatment of any central nervous system (CNS) cancer. More than 95% of selective damage of SJGBM2 glioma cells was observed at 1 μM of CN-GM conjugate with almost 100% viability of non-cancerous HEK293 cells, although this ability was diminished at lower concentrations. However, further conjugation of Tf to obtain CN-GM-Tf allowed the achievement of selective targeting and prominent anti-cancer activity at a 100-fold lower concentration of 10 nM. Furthermore, both conjugates were capable of effectively damaging several other brain tumor cells, which were not well responsive towards the single treatment of GM. The capability of BBB penetration of the conjugates was observed using a zebrafish model, which confirms the CNDs' competence as an excellent nanocarrier to the CNS.

The development of a gold nanoparticle aptamer assay is persued for rapid and sensitive determination of histamine in foodstuffs, which could be deployed for on-site use. The assay is based on a histamine-specific aptamer and gold nanoparticles and the salt-induced aggregation of the particles in the presence of histamine indicated by the color change from red to blue. Gold nanoparticle size, salt type, and concentration as well as aptamer concentration were optimized, and using optimum conditions, a limit of detection of 8 nM (~ 0.05 mg/kg) was obtained. Finally, the aptamer AuNP assay was applied to the determination of histamine in quality control fish samples. The histamine levels of these samples had previously been determined using HPLC and commercial ELISA kits by numerous independent laboratories and a good correlation was obtained. The developed AuNP assay is rapid, sensitive, and reproducible. Graphical abstract.

A sensitive and selective differential pulse-adsorptive anodic stripping voltammetry (DP ASV) method has been developed for trace determination of timolol maleate drug in complex matrices. The established method is first of its kind for timolol maleate (TM) determination with a wide linear dynamic range (1.0 × 10−9–2.0 × 10−5 mol L−1) coupled with limits of detection and quantification of 7.1 × 10−10 mol L−1 (0.31 μg L−1) and 2.4 × 10−9 mol L−1 (1.04 μg L−1), respectively. A relative standard deviation (±0.565%, n = 5) at 2.0 × 10−6 mol L−1 concentration of the drug was obtained. The method was validated by comparison with standard HPLC method. Statistical treatment of data using Student t and F tests at P = 0.05 revealed no significant differences between experimental and tabulated t and F values. Due to high selectivity towards TM and minimum interference from common organic and inorganic compounds the developed probe was successfully applied for trace analysis of TM in an eye drop, urine and water samples. The established method has a unique advantage to be quantitatively applied to a variety of samples due to rapid response, short analytical time and high sensitivity, and excellent selectivity with good reproducibility. The figures of merits were compared successfully with some reported electrochemical, chromatographic and spectrochemical methods. The interfacial properties of the modified Nafion/c-MWCNTs/GCE were explored by electrochemical impedance spectroscopy (EIS).

High-risk pathogens such as Francisella tularensis and Yersinia pestis are categorized as highly hazardous organisms that can be used as biological weapons. Given the extreme infectivity of these potential biowarfare agents, a rapid, sensitive, cost-effective, and specific method for their detection is required. Here, we report the multiplexed amplification detection of genomic DNA from Francisella tularensis and Yersinia pestis. Amplification was achieved using isothermal recombinase polymerase amplification, exploiting tailed primers, followed by detection using a nucleic-acid lateral flow assay. Excess primers were removed using a novel fishing strategy, avoiding the use of postamplification purification that requires centrifugation and infers additional assay cost. The entire assay is completed in less than 1 h, achieving limits of detection of 243 fg (1.21 × 102 genome equivalent) and 4 fg (0.85 genome equivalent) for Francisella tularensis and Yersinia pestis, respectively.

Trichomoniasis, caused by Trichomonas vaginalis, is the leading nonviral sexually transmitted infection worldwide. We report the selection of a DNA aptamer against a T. vaginalis adhesion protein, AP65, using a microtiter plate-based in vitro combinatorial chemistry process termed systematic evolution of ligands by exponential enrichment. The enriched library pool was sequenced by next-generation sequencing, and several aptamer candidates with high affinity and specificity were identified. The aptamer with the highest affinity and specificity had a KD in the low nanomolar range, as confirmed by three different techniques: surface plasmon resonance, enzyme-linked aptamer assay, and biolayer interferometry. The selected aptamer was demonstrated to have a high specificity to the AP65 protein and to T. vaginalis cells with no cross-reactivity to other enteric and urogenital microorganisms. Current work is focused on the development of inexpensive and easy-to-use aptamer-based diagnostic assays for the reliabl...

The interface of nucleic acids and nanomaterials is among the most promising fields in recent years. Considerable efforts have been devoted to the development of novel systems based on the two components for various promising applications such as sensing, bioimaging, drug delivery, and theranostics. However, the determination of nucleic acid concentration in these systems remains as a challenge due to the interference of nanoparticles. To this end, we developed a simple, yet reliable, method to quantify the nucleic acid concentration in their nanoparticle or polymer conjugates based on circular dichroism (CD) spectroscopy. In this paper, three nucleic acids, namely, DNA sodium salt from calf thymus (NaDNA), DNA from herring sperm (hsDNA), and ribonucleic acid from torula yeast (tyRNA), were noncovalently conjugated to three nanoparticles. The concentrations of the three nucleic acids in their nanoparticle conjugates were successfully determined on the basis of CD spectra calibration curves.

The use of lateral flow assays exploiting antibodies is well established in different fields due to their advantages, which include low cost, ease of production and rapid response, with the only required end-user intervention being sample addition. In recent years, aptamer-based lateral flow assays are garnering increasing interest offering a highly cost-effective and more flexible alternative to antibodies. In this review, an overview of the aptamer-based lateral flow assays developed to date is provided, highlighting the advantages of using aptamers and their ability to be incorporated into formats not possible with antibodies.

In this work we report the mapping of the binding site of the only progesterone aptamer published to date, in an approach referred to as aptatope mapping. By linking the binding data obtained from microscale thermophoresis analysis to the structural differences on the ring structure of a range of steroids, we elucidated the moieties involved in aptamer-progesterone binding. This approach can be further exploited for the characterization of aptamer specificity and ultimately facilitate the development of aptamer-based assays depending on the desired specificity.

Carbon dots, a recent member of the renowned carbon-based nanomaterials family, have attracted significant interest from various fields. The extraordinary properties, including excellent photoluminescence, high biocompatibility, and tunable surface functionalities as well as the abundant and inexpensive nature, have led to remarkable bioapplications in bioimaging, drug delivery, and theranostics development. In this article, studies on the interaction of C-dots with biomacromolecules are reviewed first, and recent developments of C-dots for target-specific bioimaging, drug delivery as well as theranostics development are highlighted and discussed.

A simple strategy has been developed for determination and speciation of bismuth(III) & (V) coupled dual wave β-correction spectrophotometry. The principle of the study was based on the reaction of bismuth(III) with reagent 1-( o -arsonophenylazo)-2-napthol-3,6-disulphuric acid (Thorin-I) to form a deep red orange colored complex species. The absorbance intensity was recorded at λ max = 540 nm. The method exhibits a wide linear range (0.25–20.0 μg mL − 1 ) and Ringbom's plots in the range 1.6–10.0 μg mL − 1 for bismuth(III) ions, respectively. The lower limit of detection (LOD) and quantification (LOQ) obtained were 0.045 and 0.15 μg mL − 1 , respectively. Bismuth(V) was determined after reduction to bismuth(III) with Mn 2 + in acidic solution. Trace level speciation studies were performed with satisfactorily recovery percentages for bismuth species. The method was applied for analysis of total bismuth in tap water, well water, marine water, hair sample, pharmaceutical formulations (cream ) and in certified reference material (IAEA Soil-7). The results were compared successfully in terms of F and t- test with the reference method inductively coupled plasma-mass spectrometry(ICP-OES) and no significant difference was noticed at 95% confidence ( P = 0.05). The developed method has a unique advantage to be quantitatively applied to a variety of samples due to rapid response, short analytical time and ability to be detected by naked eye with good reproducibility. The LOD offered has a value lower than that recommended by World Health Organization (WHO) and compared successfully with some reported spectrochemical and electrochemical methods.

Anabolic androgenic steroids (AAS) are frequently abused in human and animal sports as performance-enhancing drugs, and consequently their use is controlled by international sports authorities. Testosterone is one of the most frequently used AAS, and therefore the accurate determination of its levels in biological fluids is very important. The authors describe the selection of testosterone-binding aptamers performed using a classic SELEX approach with the target immobilized on magnetic beads. Counter selections with structurally similar steroids were implemented at different stages. Pools from different selection rounds were sequenced with Next Generation Sequencing and ten aptamer candidates were selected for further characterization. Low nanomolar range dissociation constants were calculated by a bead-based PCR assay and verified by microscale thermophoresis. Future work will focus on the development of aptamer-based platforms for the sensitive detection of testosterone in biological samples and the validation of these assays for the rapid screening of suspicious samples.

This study investigates the surface chemistry properties of the tyrosinase enzyme Langmuir monolayer at air-aqueous interface using sodium chloride in the subphase to induce the surface activity of the enzyme. Investigation of surface packing and stability of the tyrosinase Langmuir monolayer were performed using surface chemistry experiments while spectroscopic analysis was done to study enzyme conformation. It was found that the tyrosinase enzyme forms a fluid film at air-aqueous interface with good stability as shown by compression-decompression cycles experiments and stability measurements at various surface pressures. UV-vis absorption and fluorescence measurements at different surface pressures revealed that the Langmuir monolayer has good homogeneity with no evidence of aggregates during compression. To gain insight on the conformation of tyrosinase Langmuir monolayer p-polarized infrared-reflection absorption spectroscopy was used. It was found that at high surface pressures the predominant secondary structures were β-sheets while at lower surface pressure both α -helices and β-sheets were present. The circular dichroism spectra were obtained by transferring the Langmuir monolayer at 10 mN.m−1 to a solid quartz support (Langmuir-Blodgett film, LB film), which showed that the major conformation present were α-helices. Images from the immobilized LB films were obtained using atomic force microscopy which showed homogenous and regular deposition with a mean thickness ranging from 3 to 4 nm.

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays ( KD of 3-34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

Alpha-l-fucosidase is a known biomarker for hepatocellular carcinoma that has shown great potential in diagnostics. Most of the focus for this enzyme has been on the free form found in serum; however, little is known of the properties of the minor portion of membrane-bound alpha-l-fucosidase. To better understand the properties of membrane-bound alpha-l-fucosidase, this enzyme was surveyed at the air-water interface. Alpha-l-fucosidase is able to form a stable Langmuir monolayer, which was confirmed through surface-pressure and surface-potential area isotherms, as well as infrared reflection-absorption spectroscopy (IRRAS). Furthermore, an interaction between the alpha-l-fucosidase Langmuir monolayer and a specific antibody for this enzyme, FUCA2, was observed.

A simple procedure for dispersive liquid-liquid microextraction (DLLME) of Cr(III) and Cr(VI) species has been established. The ion associate represented by halochromate (CrO 3 Cl − ) anion and rhodamine 6G hydrochloride dye (RG + ) cation is selectively extracted via DLLME following micro-volume UV–Vis spectrometric detection. The reaction conditions as pH adjustment, acidity, concentration of reagent and electrolyte were optimized. At appropriate conditions, the proposed method exhibited a two order of magnitude wide linear range of 25–950 μg L − 1 with a detection limit of 7.48 μg L − 1 for Cr(VI). The relative standard deviation of 2.16 ( n = 5) at 90 μg L − 1 of Cr(VI) was evaluated. The ions that commonly occur in real water samples were also investigated and pose no interference to Cr(VI) determination. Regarding Cr(III) species, it was evaluated after oxidation to Cr(VI) with H 2 O 2 in KOH media. The method was validated by determination of Cr(III) and Cr(VI) in spiked water samples (tap and sea water). The results were also compared successfully with the data obtained by inductively coupled plasma-optical emission spectrometry (ICP-OES). The difference in accuracy and precision (Student t - and F- tests) between the two methods was insignificant at 95% confidence. Thermodynamic parameters (Δ H , Δ S , and Δ G ) of the produced complex ion associate were determined.

In the past few years, we have seen intense interest grow in chemistry, toxicity and analysis of biogenic amines (BAs). Thus, a comprehensive review on clean-up and recent advances in analysis of biogenic amines (BAs) in human body and dairy products of foodstuffs are presented. Liquid-liquid extraction, solid phase extraction, solid phase microextraction, dispersive liquid-liquid microextraction (DLLME), cloud point extraction and hollow fiber-liquid phase microextraction represent the most common preconcentration techniques for BAs. HPLC, GC, TLC, spectrofluorimetry, capillary zone electrophoresis coupled with mass spectrometry are the most common analytical techniques used for analysis of BAs. DLLME techniques offer benefits over centrifugation, filtration and solid-phase extraction. The milestones and combination of nanotechniques in the DLLMEs field and green aspects of BAs in literature; advantages and drawbacks are addressed. A major focus on analysis of BAs revealed no use of coupling DLLME techniques with electroanalytical techniques in particular stripping voltammetry. Thus, voltammetric techniques at surface modified electrodes implemented with DLLME techniques is highly recommended for developing low cost and precise methods for analysis of BAs at ultra trace levels in foodstuffs. Conclusions have been drawn for future research is proposed.

A series of π-conjugated nonlinear 4′-tricyanovinylhydrazone (TCH) dyes was synthesized and characterized. The redox behaviors of the TCH dyes were studied by direct current (DC), and derivative polarography (DP), cyclic voltammetry (CV) and controlled potential coulometry (CPC). Excluding the nitro derivatives, the DC and DP polarograms of the compounds at pH < 5 showed two irreversible waves (peaks) corresponding to reduction of the CC– and the hydrazone (CN–) groups. The DC and DP polarography of the nitro derivatives at pH < 5 showed three waves (peaks). Plots of cathodic peak current (ip,c) vs. square root of scan rate (ν1/2) and log ip,c vs. log ν at −0.59 and −0.93 V at pH 4.46 were linear. The kinetic parameters (log kof,h, ΔG* and D) of the compounds were determined. Poor correlations for substituents on the E1/2 of the dyes with the Hammett substituent constants were noticed. Based on the chromatographic separation, spectroscopic characterization and molecular weight determination of the end products of the electrolyzed species of 4′-nitrotricyanovinylhydrazone, an electrochemical reduction mechanism is proposed.

A micro-volume spectrofluorimetric coupled ultrasound-assisted ion association dispersive liquid–liquid microextraction (USA-IA-DLLME) procedure for the total determination and speciation of chromium(III & VI) species has been established. This demonstration was based upon the formation of a complex ion associate between halochromate (CrO3Cl−) and rhodamine 6G hydrochloride dye (RG+). At optimal conditions, the method exhibited a three order of magnitude wide linear concentration range (1.0–1000 μg L−1), with detection and quantification limits of 0.57 μg L−1 and 1.9 μg L−1, respectively. Competent cations, anions and oxoanions did not interfere with the chromium(VI) determination. Chromium(III) was also evaluated after its conversion to chromate with H2O2 in alkaline media. The nature of the extractant and the disperser, their volumes and the extraction time were optimized. The fluorescence quenching mechanism of the complex ion associate was discussed. The method was validated by the determination of chromium(VI) in real water (sea and tap water) samples. The results were also compared successfully with those from inductively coupled plasma-optical emission spectrometry (ICP-OES) in terms of the student’s t- and F test data at 95% confidence.

Because accidents, disease and aging compromise the structural and physiological functions of bones, the development of an in vivo bone imaging test is critical to identify, detect and diagnose bone related development and dysfunctions. Recent advances in fluorescence instrumentation offer a new alternative for traditional bone imaging methods. However, the development of new in vivo bone imaging fluorescence materials has significantly lagged behind. Here we show that carbon dot nanoparticles (C-dots) with low quantum yield ("dark") bind to calcified bone structures of live zebrafish larvae with high affinity and selectivity. Binding resulted in a strong enhancement of luminescence that was not observed in other tissues, including non-calcified endochondral elements. Retention of C-dots by bones was very stable, long lasting, and with no detectable toxicity. Furthermore, we found C-dots to be a suitable carrier to deliver fluorescein to bones. These observations support a novel and revolutionary use of C-dots as highly specific bioagents for bone imaging and diagnosis, and as bone-specific drug delivery vehicles.

The current study explores the use of quercetin for developing a highly selective spectrofluorimetric methodology for trace determination, speciation and thermodynamic characterization of tungstate (WO42−) species in water. The study relies on the principle of chelate formation between WO42− and quercetin with subsequent increase in the emission intensity. The developed method could be applied successfully in a wide linear range (1.0–400.0 μg L−1) with a detection limit of 0.28 μg L−1 and quantification limit of 0.92 μg L−1 at λex/em = 400/492 nm. The developed method was successfully applied in real tap and waste water samples. The suitability of the proposed method was further validated by inductively coupled plasma-optical emission spectrometry (ICP-OES) in terms of student's t and F tests at 95% confidence. Characterization (NMR, FTIR and electronic spectra), stoichiometry, stability constant, fluorescence mechanism and thermodynamic parameters (ΔH, ΔS, and ΔG) of the produced complex species were evaluated and properly assigned. The fluorescence quenching mechanism of tungstate quercetin complex by Triton X-100 was also evaluated for computing Stern-Volmer quenching constant and approximating quenching sphere. The method showed a clear significance over most of the reported methods for tungsten in literature in terms of good accuracy, robustness, ruggedness, short analytical time and cost-effectiveness.

Dispersive liquid-liquid microextraction (DLLME) techniques have attracted considerable interest because they are cost effective, easy to operate, and reliably preconcentrate trace levels of analytes in complex matrices. This comprehensive review is concerned with principles, applications and developments of DLLME techniques for analysis of trace emerging pesticides in water. DLLME techniques have had few couplings to spectrofluorimetric methods and relatively none with electrochemical techniques. We highly recommend thin-layer stripping voltammetric techniques at surface-modified electrodes and spectrofluorimetric techniques coupled and implemented with DLLME. Great attention should be focused on developing low-cost, precise methods for analysis of trace concentrations of pesticides in various biological and environmental samples. We describe milestones and the combination of nanotechniques in the DLLME field, green aspects, advantages and shortcomings of known DLLME protocols.

The sorption profile of trace concentrations of bismuth(III) species from aqueous KI–H 2 SO 4 media onto the ion pairing procaine hydrochloride (PQ + .Cl − ) based polyurethane foam (PUFs) was studied. A dual–mode of bismuth(III) sorption as a ternary complex ion associate [PQ + .BiI 4 − ] involving absorption due to a “weak base anion exchanger” and an added component for “surface adsorption” seems the proposed mechanism for bismuth(III) retention. The capacity of bismuth(III) sorption was found equal 40.05 ± 1.10 mg g −1 . Complete separation of spiked bismuth(III) at various concentrations (5–15 μg mL −1 ) from water onto the proposed sorbent packed columns at 5 mL min −1 flow rate was successfully achieved. The retained bismuth(III) species were recovered quantitatively from the sorbent packed columns (98.4 ± 2.4%, n = 5) with HNO 3 (1.0 mol L −1 ). Bismuth(V) after reduction to Bi(III) was also preconcentrated, recovered from the sorbent packed column and subsequently analyzed by inductively coupled plasma–optical emission spectrometry (ICP–OES). The height equivalent to the theoretical plates (HETP), number of plates (N), critical and breakthrough capacities of sorbent packed column towards bismuth(III) retention were evaluated. Based on these results, a simple and sensitive PQ + .Cl − –PUF packed column was developed for determination and speciation of trace concentrations of bismuth(III & V) species in water by ICP–OES. The limits of detection (LOD) and quantification (LOQ) of Bi(III) were found 0.09 and 0.30 μg L −1 , respectively. The relative standard deviation (RSD) for 5 replicates of 50 ng L −1 Bi at 2.0 mL min −1 flow was ±3.7%. The proposed column was validated and applied for the preconcentration, separation and subsequent determination of analyte in Environmental water reference material (TMDW) and water samples. The results were found to be in good agreement with the CRM at the 95% confidence level.

A fast and selective method for cadmium(II) removal from water by procaine hydrochloride (PQ+·Cl−) immobilized polyurethane foam (PUFs) sorbent was developed. The method was based upon formation of [CdI4]−2aq in the test aqueous KI solution and subsequent extraction by PQ+·Cl− treated PUFs. The sorption of Cd2+ ions followed first order equation with an overall rate constant of 0.132 ± 0.033 min−1. The values of ΔH and ΔS were −41.54 ± 0.9 kJ mol−1 and −144.58 ± 3.1 J mol−1 K−1, respectively with a correlation factor of 0.998. The thermodynamic parameters (ΔH, ΔS and ΔG) suggest that the sorption was spontaneous and exothermic process. The negative value of ΔS provides indication of moderate sorption of [CdI4]2− ion associate and ordering of the ionic charges without compensatory disordering of the sorbed species onto the sorbent. Cadmium(II) sorption is mainly dominated by absorption related to “solvent extraction” and an added component for “surface adsorption”. PQ+·Cl− treated PUFs packed column was also tested for preconcentration of trace concentrations of cadmium(II) species in various water samples. The retained Cd species were successfully recovered with dilute HNO3 (1.0 mol L−1) and subsequently analyzed by ICP–OES. Thus, the proposed sorbent packed column provides efficient removal of traces of cadmium(II) ions from water samples. The method could be extended for preconcentrate of trace and ultra trace cadmium species from large samples onto PUF packed column and subsequently analyzed. Indeed, the developed method could be satisfactorily applied to the determination of trace Cd ions in natural water.

Detection of alpha-l-fucosidase has been shown to have relevance in diagnosing hepatocellular carcinoma. Few assays have been developed to measure this enzyme, with most relying on colorimetric techniques involving the enzyme’s kinetics. While these assays are facile and quick, the sensitivity is not always sufficient for early tumor detection. To improve upon previous assays for alpha-l-fucosidase, a fluorescence based immunoassay was produced implementing an alpha-l-fucosidase specific antibody (FUCA2). The immobilization of the alpha-l-fucosidase-specific antibody onto a quartz slide was investigated with several bioconjugation approaches and an immunoassay for detection of alpha-l-fucosidase was produced. The immunoassay was utilized to produce calibration curves for quantifying alpha-l-fucosidase concentrations in both PBS and human blood serum. A detection limit of 10 nM was found using human blood serum, which is well below the diagnostic cutoff point of 80 nM.

The application of nanoparticles (NPs) for biomedical use is one of the fastest growing fields in science. The great potential has attracted attention from many fields, including chemistry, biology, medicine, and engineering. However, with relatively little understanding of biomacromolecule–NP interactions, there are increasing safety concerns about the use of nanomaterials in biomedical fields. To determine the biocompatibility of NPs and evaluate their nanosafety, great efforts are currently being made on investigating biomacromolecule–NP interactions. Spectroscopic methods and strategies play an important role in these investigations to help us understand the mechanistic basis for the biological activity of NPs, which is essential for the safe application of nanotechnology. Herein, in this chapter, we summarize the recent progress in the study of protein–NP, DNA–NP, and lipid–NP interactions based on various spectroscopic techniques.

The sorption profile of cadmium (II) ions from aqueous iodide media onto procaine hydrochloride (PQ+·Cl−) treated polyurethane foams (PUFs) solid sorbent was studied. PQ+·Cl− treated PUFs solid sorbent was found suitable and fast for Cd2+ uptake as [CdI4]aq2−. Thus, removal of Cd2+ at trace levels by the sorbent packed columns was achieved. The sorbed Cd2+ species onto packed column were recovered with HNO3 (10.0 mL, 1.0 mol L−1) prior determination by inductively coupled plasma-optical emission spectrometry (ICP-OES). Plot of Cd2+ ions concentration was linear in the range 0.05–15 μg L−1. The limits of detection and quantification of Cd2+ were found 0.01 μg L−1 and 0.033 μg L−1, respectively. Such limits could be improved to lower values by retention of Cd2+ species from large sample volumes of the aqueous phase at the optimized conditions. The relative standard deviation of the packed column for the extraction and recovery of standard aqueous solutions (0.1 L) containing 1.0 and 5.0 μg L−1 (n = 3) of Cd2+ ions at flow rate of 5.0 mL min−1 were 1.98 and 2.9%, respectively. The method was validated by analysis of Cd in certified reference materials (CRMs) IAEA-Soil-7 and TMDW water and wastewater samples.

The redox behavior of a palladium(II)-3,4-bis(2-hydroxyphenyl-imino) cyclobut-1-en-1,2-diol complex at Pt, Au and hanging mercury drop electrodes (HMDE) was studied for developing a low cost and precise method for determination of Pd concentration in road dust and other environmental samples. Hence, a controlled adsorptive accumulation of this complex on HMDE provided the basis for adsorptive cathodic stripping voltammetric (AdCSV) measurements of palladium at nanomolar levels at pH 9–10 and at −0.64 V vs. the Ag/AgCl reference electrode. The calibration plot was obtained in the range of 1.87 × 10−9 to 3.05 × 10−7 M (0.2 to 32.5 μg L−1) Pd. The limit of detection was found to be 4.70 × 10−10 M (0.05 μg L−1), with a relative standard deviation (RSD) of ±2.1% (n = 5) at 2.0 μg L−1 Pd level. Common anions and cations did not interfere in the determination of Pd concentration. The method was applied to the determination of the concentration of Pd in pure authentic samples, roadside dust and water samples. The method offers a simple system coupled with good reproducibility, accuracy, ruggedness and cost effectiveness.

The redox behavior of spironolactone (SP) drug in BrittonâRobinson (BR) buffer of pH 2â11 was investigated by differential pulse cathodic stripping voltammetry (DPCSV) and cyclic voltammetry (CV) at hanging mercury dropping electrode (HMDE). At pH 9â10.5, the DPCSV of SP drug showed two cathodic peaks at â1.15 and â1.38 V at the HMDE vs. Ag/AgCl reference electrode. In the CV, at pH 9â10, the dependence of the cathodic peak current, Ip,c and peak potential, Ep,c of the second peak (Ep,c2) on the scan rate (ν) and on the depolizer (SP) concentrations was typical of an electrode coupled (EC) chemical reaction type mechanism. The plot of Ip,c at â1.380 V of the DPCSV vs. SP concentration at pH 9 was linear over the concentration range of 1.2Ã10â10â9.6Ã10â7 M. The lower limit of detection (LLOD) and limit of quantification (LOQ) of the drug were 1.1Ã10â11 and 4.14Ã10â11 M, respectively. The method was successfully applied for the analysis of SP residues in industrial wastewater, in pure form (98.2±3.1%) and in drug formulations e.g. Aldactone® tablet (98.35±2.9%).The method was validated by comparison with HPLC and the official data methods. Keywords: Spironolactone drug residue, Cathodic stripping voltammetry, Wastewater, Aldactone® tablets, Electrode mechanism

A simple new micelle mediated preconcentration method was developed for analysis of sunset yellow (SY) prior to its spectrophotometric determination. The method was based upon cloud point extraction of the ion associate of SY and trioctylamine (TOA) in HCl–Triton X-100. In the surfactant phase the SY species react with TOA yielding hydrophobic ion associate of SY − ·TOA + . The distribution coefficient of SY between surfactant-rich phase and aqueous phase was approximately 104. Validation was tested by comparing the results with standard HPLC. Isotherm and thermodynamic parameters, chemical equilibrium, extraction constants and stiochiometry of the associate were assigned.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

In the past few years, we have seen intense interest grow in chemistry, toxicity and analysis of biogenic amines (BAs). Thus, a comprehensive review on clean-up and recent advances in analysis of biogenic amines (BAs) in human body and dairy products of foodstuffs are presented. Liquid-liquid extraction, solid phase extraction, solid phase microextraction, dispersive liquid-liquid microextraction (DLLME), cloud point extraction and hollow fiber-liquid phase microextraction represent the most common preconcentration techniques for BAs. HPLC, GC, TLC, spectrofluorimetry, capillary zone electrophoresis coupled with mass spectrometry are the most common analytical techniques used for analysis of BAs. DLLME techniques offer benefits over centrifugation, filtration and solid-phase extraction. The milestones and combination of nanotechniques in the DLLMEs field and green aspects of BAs in literature; advantages and drawbacks are addressed. A major focus on analysis of BAs revealed no use of coupling DLLME techniques with electroanalytical techniques in particular stripping voltammetry. Thus, voltammetric techniques at surface modified electrodes implemented with DLLME techniques is highly recommended for developing low cost and precise methods for analysis of BAs at ultra trace levels in foodstuffs. Conclusions have been drawn for future research is proposed.

Lupin is increasingly being used in a variety of food products due to its nutritional, functional and nutraceutical properties. However, several examples of severe and even fatal food-associated anaphylaxis due to lupin inhalation or ingestion have been reported, resulting in the lupin subunit β-conglutin, being defined as the Lup an 1 allergen by the International Union of Immunological Societies (IUIS) in 2008. Here, we report an innovative method termed aptamer-recombinase polymerase amplification (Apta-RPA) exploiting the affinity and specificity of a DNA aptamer selected against the anaphylactic β-conglutin allergen termed β-conglutin binding aptamer II (β-CBA II), facilitating ultrasensitive detection via isothermal amplification. Combining magnetic beads as the solid phase with Apta-RPA detection, the total assay time was reduced from 210 min to just 25 min, with a limit of detection of 3.5 × 10−11 M, demonstrating a rapid and ultrasensitive generic methodology that can be used with any aptamer. Future work will focus on further simplification of the assay to a lateral flow format.

The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17β-estradiol as a model system. Microscale thermophoresis, apta-PCR affinity assay and surface plasmon resonance were explored to evaluate the reported aptamers' binding properties in terms of affinity and specificity, and were demonstrated to be successfully applied to the analysis of aptamer-small molecule interactions.

Peptides and proteins have become very promising drug candidates in recent decades due to their unique properties. However, the application of these drugs has been limited by their high enzymatic susceptibility, low membrane permeability and poor bioavailability when administered orally. Considerable efforts have been made to design and develop drug delivery systems that could transport peptides and proteins to targeted area. Although it is of great importance to determine the composition after loading a drug to the carrier, the ability to do so is significantly limited by current analytical methods. In this letter, five important proteins, α1-antitrypsin, hemoglobin human, human serum albumin, human transferrin and r-globulin were chemically conjugated to two model drug carriers, namely carbon dots and polymer O-(2-carboxyethyl) polyethylene glycol. A simple yet convenient method based on circular dichroism spectroscopy was developed to determine the compositions of the various protein-carrier conjugates.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.