Quantitative Colorimetric Sensing of Carbidopa in Anti-Parkinson Drugs Based on Selective Reaction with Indole-3-Carbaldehyde.

The quality of life of patients affected by Parkinson's disease is improved by medications containing levodopa and carbidopa, restoring the dopamine concentration in the brain. Accordingly, the affordable quality control of such pharmaceuticals is very important. Here is reported the simple and inexpensive colorimetric quantification of carbidopa in anti-Parkinson drugs by the selective condensation reaction between the hydrazine group from carbidopa and the formyl functional group of selected aldehydes in acidified hydroalcoholic solution. An optical assay was developed by using indole-3-carbaldehyde (I3A) giving a yellow aldazine in EtOH:H2O 1:1 (λmax~415 nm) at 70 °C for 4 h, as confirmed by LC-MS analysis. A filter-based plate reader was used for colorimetric data acquisition, providing superior results in terms of analytical performances for I3A, with a sensitivity ~50 L g-1 and LOD ~0.1 mg L-1 in comparison to a previous study based on vanillin, giving, for the same figures of merit values, about 13 L g-1 and 0.2-0.3 mg L-1, respectively. The calibration curves for the standard solution and drugs were almost superimposable, therefore excluding interference from the excipients and additives, with very good reproducibility (avRSD% 2-4%) within the linear dynamic range (10 mg L-1-50 mg L-1).

Serotonin-Derived Fluorophore: A Novel Fluorescent Biomaterial for Copper Detection in Urine.

We took advantage of the fluorescent features of a serotonin-derived fluorophore to develop a simple and low-cost assay for copper in urine. The quenching-based fluorescence assay linearly responds within the concentration range of clinical interest in buffer and in artificial urine, showing very good reproducibility (CVav% = 4% and 3%) and low detection limits (16 ± 1 µg L-1 and 23 ± 1 µg L-1). The Cu2+ content was also estimated in human urine samples, showing excellent analytical performances (CVav% = 1%), with a limit of detection of 59 ± 3 µg L-1 and a limit of quantification of 97 ± 11 µg L-1, which are below the reference value for a pathological Cu2+ concentration. The assay was successfully validated through mass spectrometry measurements. To the best of our knowledge, this is the first example of copper ion detection exploiting the fluorescence quenching of a biopolymer, offering a potential diagnostic tool for copper-dependent diseases.

Sensing of Catecholamine in Human Urine Using a Simple Colorimetric Assay Based on Direct Melanochrome and Indolequinone Formation.

We used the first enzyme-free synthesis and stabilization of soluble melanochrome (MC) and 5,6-indolequinone (IQ) derived from levodopa (LD), dopamine (DA), and norepinephrine (NE) oxidation to develop a simple colorimetric assay for catecholamine detection in human urine, also elucidating the time-dependent formation and molecular weight of MC and IQ using UV-Vis spectroscopy and mass spectrometry. The quantitative detection of LD and DA was achieved in human urine using MC as a selective colorimetric reporter to demonstrate the potential assay applicability in a matrix of interest in therapeutic drug monitoring (TDM) and in clinical chemistry. The assay showed a linear dynamic range between 5.0 mg L-1 and 50.0 mg L-1, covering the concentration range of DA and LD found in urine samples from, e.g., Parkinson's patients undergoing LD-based pharmacological therapy. The data reproducibility in the real matrix was very good within this concentration range (RSDav% 3.7% and 6.1% for DA and LD, respectively), also showing very good analytical performances with the limits of detection of 3.69 ± 0.17 mg L-1 and 2.51 ± 0.08 mg L-1 for DA and LD, respectively, thus paving the way for the effective and non-invasive monitoring of dopamine and levodopa in urine from patients during TDM in Parkinson's disease.

Conformationally Constrained Sialyl Analogues as New Potential Binders of h-CD22.

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.

Colorimetric determination of carbidopa in anti-Parkinson drugs based on 4-hydroxy-3-methoxybenzaldazine formation by reaction with vanillin.

In this paper is reported the selective colorimetric detection and quantification of carbidopa, an inhibitor of aromatic amino acid decarboxylase, in the co-presence of levodopa as dopamine precursor in pharmaceutical formulations for the treatment of Parkinson's disease. The method is based on the selective condensation reaction between the hydrazine group from carbidopa and the formyl functional group of vanillin, a natural flavoring agent, in acidified alcoholic solution. The yellow color development (λmax ~ 420 nm) due to the formation of 4-hydroxy-3-methoxybenzaldazine (HMOB) was observed for carbidopa only, whereas levodopa, lacking the hydrazine group, did not color the solution, as expected. The calibration curves for two tablet formulations of levodopa in combination with carbidopa (4:1) were superimposable with levodopa/carbidopa (4:1), as well as carbidopa alone, in standard solution, i.e., the excipients and additives did not interfere with carbidopa determination, corresponding to a mean recovery about 105%. The linear dynamic range was between 5.00 and 50.0 mg L-1 with very good reproducibility within this range (CVav% about 3-4%) and very good sensitivity, with limits of quantification of about 1 mg L-1. The colorimetric method developed here is very simple, inexpensive, and effective for drug estimation and quality control of pharmaceutical formulations.

Melanochrome-based colorimetric assay for quantitative detection of levodopa in co-presence of carbidopa and its application to relevant anti-Parkinson drugs.

In this paper is reported the selective detection and quantification of levodopa in co-presence of carbidopa. The method took advantage of the spontaneous oxidation and color development of levodopa at basic pH here driven by alkaline earth cations and co-solvent in solution. We have shown for the first time the generation and stabilization of the purple melanochrome from levodopa, by using magnesium acetate and dimethyl sulfoxide, which was here exploited for the development of a quantitative colorimetric assay for the active principle ingredient in commercial drugs for the treatment of Parkinson's disease. The calibration curves of levodopa in the two tablet formulations, containing carbidopa as decarboxylase inhibitor, showed a common linear trend between 10 mg L-1 and 40 mg L-1 with levodopa alone or in combination with carbidopa in standard solutions, with very good reproducibility (CVav%, 3.3% for both brand and generic drug) and very good sensitivity, with limit of quantification about 0.6 mg L-1 in any case. The colorimetric method here developed is very simple and effective, appearing as a rapid and low-cost alternative to other methodologies, involving large and expensive instrumentations, for drug estimation and quality control of pharmaceutical formulations.

A biomimetic enzyme-linked immunosorbent assay (BELISA) for the analysis of gonadorelin by using molecularly imprinted polymer-coated microplates.

An original biomimetic enzyme-linked immunoassay (BELISA) to target the small peptide hormone gonadorelin is presented. This peptide has been recently listed among the substances banned in sports by the World Antidoping Agency (WADA) since its misuse by male athletes triggers testosterone increase. Hence, in response to this emerging issue in anti-doping controls, we proposed BELISA which involves the growth of a polynorepinephrine (PNE)-based molecularly imprinted polymer (MIP) directly on microwells. PNE, a polydopamine (PDA) analog, has recently displayed impressive performances when it was exploited for MIP preparation, giving even better results than PDA. Gonadorelin quantification was accomplished via a colorimetric indirect competitive bioassay involving the competition between biotinylated gonadorelin linked to the signal reporter and the unlabeled analyte. These compete for the same MIP binding sites resulting in an inverse correlation between gonadorelin concentration and the output color signal (λ = 450 nm). A detection limit of 277 pmol L-1 was achieved with very good reproducibility in standard solutions (avCV% = 4.07%) and in urine samples (avCV% = 5.24%). The selectivity of the assay resulted adequate for biological specimens and non-specific control peptides. In addition, the analytical figures of merit were successfully validated by mass spectrometry, the reference anti-doping benchtop platform for the analyte. BELISA was aimed to open real perspectives for PNE-based MIPs as alternatives to antibodies, especially when the target analyte is a poorly or non-immunogenic small molecule, such as gonadorelin. Biomimetic enzyme-linked immunosorbent assay (BELISA).

Polydopamine-based quantitation of albuminuria for the assessment of kidney damage.

Proteinuria is considered indicative of kidney damage that can be related to various adverse outcomes. Nowadays, there is a huge demand for routine urine screening methods to assess health risks in clinical setting without expensive procedures and long pretreatment of the sample. To address this issue, a polydopamine-based colorimetric assay to determine urinary albumin concentration in real samples is proposed here. The core of this approach relies on the established competitive adsorption of polydopamine film and human serum albumin onto the polystyrene surface of ELISA plates. Herein, we investigated the influence of temperature and the Tris-HCl buffer concentration on the polydopamine film growth. The absorbance of polydopamine film, after 24 h at 25 °C, decreases with the increase of HSA concentration, allowing the selective detection of HSA down to 0.036 ± 0.001 g L-1 in untreated urine. This simple and low-cost bioanalytical assay exhibited very good reproducibility, %CVmean = 2 in human urine, and was superior in terms of analytical performances to some standard methods available on the market, especially in comparison to the Bradford assay, for early screening and assessment of kidney damage.

Protein-templated copper nanoclusters for fluorimetric determination of human serum albumin.

Copper nanoclusters (CuNCs) are attractive for their unique optical properties, providing sensitive fluorescent detection of several kinds of targets even in complex matrices. Their ability in growing on suitable protein and nucleic acid templates make CuNCs efficient optical reporters to be exploited in bioanalysis. In this work, we report the specific and sensitive determination of human serum albumin (HSA) in human serum (HS) and urine via CuNCs fluorescence. HSA is the most abundant protein in plasma, and plays a key role in the early diagnosis of serious pathological conditions such as albuminuria and albuminemia. Recently, HSA has become clinically central also as a biomarker to assess severity, progression, and prognosis of various cancers. We report the controlled and reproducible growth of CuNCs directly on the target analyte, HSA, which results in a fine dose-dependent fluorescent emission at 405 nm. The protocol is optimized in water, and then applied to serum and urine specimens, without matrix pretreatment. The method linearly responds within the whole concentration of clinical interest, with a sensitivity of 1.8 ± 0.1 × 10-3 g L-1 and 0.62 ± 0.03 × 10-3 g L-1 in serum and urine, respectively, and excellent reproducibility (CVav% ca. 3% for both). The assay is designed to have a single protocol working for both matrices, with recovery of 95% (HS) and 96% (urine). The stability of the fluorescence after CuNCs formation was tested over 3 days, displaying good results (yet higher in urine than in serum).

3,3',5,5'-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline values.

Sanitizing solutions against bacterial and viral pathogens are of utmost importance in general and, in particular, in these times of pandemic due to Sars-Cov2. They frequently consist of chlorine-based solutions, or in the direct input of a certain amount of chlorine in water supply systems and swimming pools. Colorimetry is one of the techniques used to measure the crucial persistence of chlorine in water, including household chlorine test kits commonly based on colorimetric indicators. Here, we show a simple and cheap colorimetric method based on 3,3',5,5'-tetramethylbenzidine (TMB), commonly used as chromogenic reagent for enzyme-linked immunosorbent assays. TMB is converted by chlorine to a colored molecule through a pH-dependent multi-step oxidation process where the chromaticity of TMB is directly proportional to chlorine content. This molecule offers several advantages over other commonly used reagents in terms of safety, sensitivity, and, peculiarly, hue modulation, giving rise to the detection of chlorine in water with a multi-color change of the indicator solution (transparent/blue/green/yellow). Moreover, through the appropriate setting of reaction conditions, such coloration is finely tunable to cover the range of chlorine concentration recommended by international health agencies for treatment of drinking water and swimming pools and to test homemade solutions prepared by dilution of household bleach during health emergency events such as during the current pandemic. Graphical abstract.

Polynorepinephrine: state-of-the-art and perspective applications in biosensing and molecular recognition.

The polymerization of norepinephrine, and the properties of the related polymer polynorepinephrine, started to be investigated barely 9 years ago and only few works were produced so far, mainly in materials science and medicine. An unexpectedly low relevance, especially if compared with the interest toward dopamine and polydopamine, differing from norepinephrine only for a hydroxyl group and whose properties were deeply investigated and applied to an impressive number of subject areas. We show here that in some cases, norepinephrine and dopamine monomers can be exchanged without virtually affecting the experimental results. But even more interesting, the choice of norepinephrine can positively influence the properties of the final polymer. In particular, the smoother and more hydrophilic surface of polynorepinephrine may enhance cell adhesion and proliferation, increase the activity of conjugated biomolecules, and induce higher cellular uptake of nanodrugs. Moreover, polynorepinephrine presents an additional anchoring point that can be exploited for further functionalization. Nevertheless, despite its potential for bioconjugation and molecular recognition, polynorepinephrine has not yet been considered in biosensing. Here we report our feelings in terms of perspective use of polynorepinephrine as new functional monomer for biomimetic receptor development by molecular imprinting, with application in affinity biosensing. Graphical abstracts.

Characterization of troponin T binding aptamers for an innovative enzyme-linked oligonucleotide assay (ELONA).

Early diagnosis of acute myocardial infarction (AMI) is of outmost importance to reduce the mortality rate, and cardiac troponins are considered the gold standard biomarkers of myocardial necrosis. In this scenario, the characterization of two troponin T (TnT)-binding aptamers as viable alternative to antibodies employed on clinical immunoassays is here reported for the first time. Their recognition ability was first investigated through surface plasmon resonance (SPR). Subsequently, an enzyme-linked oligonucleotide assay (ELONA) was developed on common 96-well polystyrene plates, both by direct and sandwich detection strategies for comparison. In both cases, the assay exhibits a detection ability of TnT in the range of low nanomolar but a great advantage on serum interference was obtained by using both aptamers in a sandwich format, with excellent reproducibility and recovery values. Despite the sensitivity needing to be enhanced to the low picomolar range, these results are encouraging for the development of new, low-cost, and rapid antibody-free colorimetric assays for AMI studies based on aptamer-Troponin T recognition.

Colorimetric determination of p-nitrophenol by using ELISA microwells modified with an adhesive polydopamine nanofilm containing catalytically active gold nanoparticles.

A microplate method is described for the quantification of p-nitrophenol (p-NPh) in urine samples where it can be found after exposure to certain insecticides such as methyl parathion or paraoxon. The assay is based on the use of a polydopamine (PDA) film doped with gold nanoparticles (AuNPs). The latter exerts a catalytic effect on the reduction of nitrophenols by NaBH4. PDA has adhesive properties and can be used to fix the AuNPs on several solid substrates, here ELISA polystyrene microwells. The optical and catalytic properties of different populations of AuNPs spontaneously grown on PDA films were investigated, mainly in terms of the relationship between AuNPs@PDA nanocomposite preparation and its catalytic activity and stability. The reduction of o-, m-, and p-nitrophenols by NaBH4 in aqueous solution was exploited as model study. The approach demonstrates that useful kinetic information on the catalytic effect can be obtained on 96-wells simultaneously by a conventional ELISA reader at a fixed wavelength of 415 nm. The method was successfully applied to the quantification of p-NPh in (spiked) urine samples and gave high reproducibility (RSD = 3.5%) and a 6.30 µM (836 µg/L) detection limit. Graphical abstract Schematic presentation of 96-wells preparation for optical quantification on ELISA reader of p-nitrophenol (p-NPh) catalytic reduction to p-aminophenol (p-APh), as model study, by NaBH4 and different population gold nanoparticles (AuNPs) grown on polydopamine (PDA) films attached onto polystyrene (PS) wells.

Real-Time Tau Protein Detection by Sandwich-Based Piezoelectric Biosensing: Exploring Tubulin as a Mass Enhancer.

Human tau protein is one of the most advanced and accepted biomarkers for AD and tauopathies diagnosis in general. In this work, a quartz crystal balance (QCM) immunosensor was developed for the detection of human tau protein in buffer and artificial cerebrospinal fluid (aCSF), through both direct and sandwich assays. Starting from a conventional immuno-based sandwich strategy, two monoclonal antibodies recognizing different epitopes of tau protein were used, achieving a detection limit for the direct assay in nanomolar range both in HBES-EP and aCSF. Afterward, for exploring alternative specific receptors as secondary recognition elements for tau protein biosensing, we tested tubulin and compared its behavior to a conventional secondary antibody in the sandwich assay. Tau-tubulin binding has shown an extended working range coupled to a signal improvement in comparison with the conventional secondary antibody-based approach, showing a dose-response trend at lower tau concentration than is usually investigated and closer to the physiological levels in the reference matrix for protein tau biomarker. Our results open up new and encouraging perspectives for the use of tubulin as an alternative receptor for tau protein with interesting features due to the possibility of taking advantage of its polymerization and reversible binding to this key hallmark of Alzheimer's disease.

In vitro selection of RNA aptamers against CA125 tumor marker in ovarian cancer and its study by optical biosensing.

Early identification of neoplastic diseases is essential to achieve timely therapeutic interventions and significantly reduce the mortality of patients. A well-known biomarker is the Cancer Antigen 125 (CA125) or 16 mucin (MUC 16), a glycoprotein of the human family of mucins, already used for the diagnostic and prognostic evaluation of ovarian cancer. Therefore, the detection of CA125 to now remains a promising tool in the early diagnosis of this tumor. In this paper, we describe the development of RNA aptamers that bind with high affinity the tumor antigen CA125. We performed eight cycles of selection against CA125 purified protein. The selected aptamers were cloned and sequenced and the binding properties of the most promising sequences were studied by Real Time PCR and Surface Plasmon Resonance (SPR) to evaluate their ability in targeting CA125 protein with perspective applications in aptamer-based bioassays.

Direct genotyping of C3435T single nucleotide polymorphism in unamplified human MDR1 gene using a surface plasmon resonance imaging DNA sensor.

Optical genotyping of C3435T single nucleotide polymorphisms (SNPs) in unamplified human multidrug resistance (MDR1) gene was here performed by a surface plasmon resonance imaging (SPRi) dual-targeting DNA assay, allowing its selective detection down to 0.18 fM of the whole genomic DNA. The result was achieved by the combination of the rational selection of the DNA probe and an optimized sample pretreatment (i.e., ultrasound fragmentation and thermal denaturation). Some assay developments and tunings were reported in a previously published research, but here, for the first time, the biosensor reliability and its analytical performance were directly tested on the unamplified human DNA extracted from lymphocytes. The assay resulted to be able to differentiate among all the possible genotypes of C3435T (homozygote and heterozygote) in the diluted genomic samples using a label-free approach and by bypassing the classical PCR amplification of the target sequences. Moreover, the reusability of the DNA-based chip allowed up to 40 subsequent measuring cycles, opening new horizons in multi-SNP genotyping based on cheap and daily routine clinical monitoring by optical biosensing.

Surface plasmon resonance applications in clinical analysis.

In the last 20 years, surface plasmon resonance (SPR) and its advancement with imaging (SPRi) emerged as a suitable and reliable platform in clinical analysis for label-free, sensitive, and real-time monitoring of biomolecular interactions. Thus, we report in this review the state of the art of clinical target detection with SPR-based biosensors in complex matrices (e.g., serum, saliva, blood, and urine) as well as in standard solution when innovative approaches or advanced instrumentations were employed for improved detection. The principles of SPR-based biosensors are summarized first, focusing on the physical properties of the transducer, on the assays design, on the immobilization chemistry, and on new trends for implementing system analytical performances (e.g., coupling with nanoparticles (NPs). Then we critically review the detection of analytes of interest in molecular diagnostics, such as hormones (relevant also for anti-doping control) and biomarkers of interest in inflammatory, cancer, and heart failure diseases. Antibody detection is reported in relation to immune disorder diagnostics. Subsequently, nucleic acid targets are considered for revealing genetic diseases (e.g., point mutation and single nucleotides polymorphism, SNPs) as well as new emerging clinical markers (microRNA) and for pathogen detection. Finally, examples of pathogen detection by immunosensing were also analyzed. A parallel comparison with the reference methods was duly made, indicating the progress brought about by SPR technologies in clinical routine analysis.

Toward nano-sized imprinted norepinephrine-derived biopolymer as artificial receptors for detecting IgG1 by surface plasmon resonance.

Bio-based nanostructured molecularly imprinted polymers (nano-MIPs), also known as 'plastibodies', have a real potential to be used as alternatives to natural antibodies. These nanostructures have recently gained significant attention for diagnostic and therapeutic purposes. In this context, we have developed polynorepinephrine (PNE)-based nano-MIPs using an eco-friendly one-pot process for the sensitive and selective detection of a model biomolecule, immunoglobulin IgG1. We first investigated non-imprinted nanostructures (nano-NIPs) based on polydopamine as reference material, using DLS, SEM, and UV-Vis spectroscopy. Subsequently, PNE scaffolds were characterized, both in the form of nano-NIPs and nano-MIPs. Concerning nano-MIPs, we used the epitope-directed imprinting technology to create binding cavities using a small peptide from the constant region of IgG1 as a template. Nano-MIPs were initially immobilized on a sensing surface to assess their binding capacity via surface plasmon resonance (SPR) spectroscopy. This strategy showed very good sensitivity, outperforming planar PNE-based imprinted films while keeping a high selectivity even in complex biological matrices such as human serum. Furthermore, we confirmed the presence of selective binding sites on nano-MIPs by flowing them, along with nano-NIPs, through a microfluidic SPR system, where they interact with the covalently immobilized analyte. This approach resulted in a good imprinting factor of 4.5. Overall, this study underscores the broad potential of these nanostructures as a viable and reusable alternative to antibodies across a variety of bioanalytical, biochemical, and immunohistochemistry analysis techniques.

CdSe/ZnS Quantum Rods (QRs) and Phenyl Boronic Acid BODIPY as Efficient Förster Resonance Energy Transfer (FRET) Donor-Acceptor Pair.

The reversibility of the covalent interaction between boronic acids and 1,2- or 1,3-diols has put the spotlight on this reaction for its potential in the development of sensors and for the fishing of bioactive glycoconjugates. In this work, we describe the investigation of this reaction for the reversible functionalization of the surface of CdSe/ZnS Quantum Rods (QRs). With this in mind, we have designed a turn-off Förster resonance energy transfer (FRET) system that ensures monitoring the extent of the reaction between the phenyl boronic residue at the meso position of a BODIPY probe and the solvent-exposed 1,2-diols on QRs' surface. The reversibility of the corresponding boronate ester under oxidant conditions has also been assessed, thus envisioning the potential sensing ability of this system.