Real-time kinetic binding studies at attomolar concentrations in solution phase using a single-stage opto-biosensing platform based upon infrared surface plasmons.

Here we present a new generic opto-bio-sensing platform combining immobilised aptamers on an infrared plasmonic sensing device generated by nano-structured thin film that demonstrates amongst the highest index spectral sensitivities of any optical fibre sensor yielding on average 3.4 × 104 nm/RIU in the aqueous index regime (with a figure of merit of 330) This offers a single stage, solution phase, atto-molar detection capability, whilst delivering real-time data for kinetic studies in water-based chemistry. The sensing platform is based upon optical fibre and has the potential to be multiplexed and used in remote sensing applications. As an example of the highly versatile capabilities of aptamer based detection using our platform, purified thrombin is detected down to 50 attomolar concentration using a volume of 1mm3 of solution without the use of any form of enhancement technique. Moreover, the device can detect nanomolar levels of thrombin in a flow cell, in the presence of 4.5% w/v albumin solution. These results are important, covering all concentrations in the human thrombin generation curve, including the problematic initial phase. Finally, selectivity is confirmed using complementary and non-complementary DNA sequences that yield performances similar to those obtained with thrombin.

[A multi-centre study of the reliability, validity and sensitivity to change of the honos65+ in psychiatry for older persons]. / Een multicenterstudie naar betrouwbaarheid, validiteit en gevoeligheid voor verandering van de honos65+ binnen de ouderenpsychiatrie.

BACKGROUND: Within the mental health care services for older persons there is a growing need for insight into and evaluation of the results of clinical treatment. The Health of the Nations Outcome Scales 65+ (honos65+) is a promising instrument for the assessment of mental, social and physical health in older persons, but it is not yet known whether it is valid for older persons in the Netherlands. AIM: To assess the reliability, validity and sensibility to change of the honos65+ when applied to older persons with psychiatric disorders. METHOD: The bio-psycho-social level of functioning of clients aged 60 and over (n=168) receiving mental health care was assessed by means of existing and validated measuring instruments and the results were compared with those obtained with the honos65+. Three months later the population sample was re-assessed in order to test the extent to which the honos65+ was sensitive to change. RESULTS: The reliability and validity of the honos65+ could be ascertained for 168 clients aged 60 and over. After three months 116 clients were re-assessed so that the sensitivity of the honos65+ to change could be noted. CONCLUSION: The honos65+ is a reliable and valid instrument for assessing clients with affective disorders such as depression and anxiety and for detecting changes in clients' problems and functioning. No conclusions could be reached regarding the reliability and validity of the honos65+ when used for clients with other psychiatric disorders because the clinical subgroups were too small for patterns to be detected.

Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads.

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads. The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430nM for thrombin was achieved. A lower detection limit for the protein (175nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45nM of thrombin demonstrating the best analytical performances. With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.

Biosensors for biomarkers in medical diagnostics.

At present, most biomarker testing is taking place at centralised dedicated laboratories using large, automated analysers, increasing waiting time and costs. Smaller, faster and cheaper devices are highly desired for replacing these time-consuming laboratory analyses and for making analytical results available at the patient's bedside (point-of-care diagnostics). Innovative biosensor-based strategies could allow biomarkers to be tested reliably in a decentralised setting, although several challenges and limitations remain, which need to be improved, in the design and application of biosensors for the appropriate interpretation of the identified and quantified biomarkers. The development of biosensors is probably one of the most promising ways to solve some of the problems concerning the increasing need to develop highly sensitive, fast and economic methods of analysis in medical diagnostics. In this review, some consideration will be given to biosensors and their application in medical diagnostics, taking into account several crucial features.

Development of an optical RNA-based aptasensor for C-reactive protein.

The development of a RNA-aptamer-based optical biosensor (aptasensor) for C-reactive protein (CRP) is reported. CRP is an important clinical biomarker; it was the first acute-phase protein to be discovered (1930) and is a sensitive systemic marker of inflammation and tissue damage. It has also a prognostic value for patients with acute coronary syndrome. The average concentration of CRP in serum is 0.8 ppm and it increases in response to a variety of inflammatory stimuli, such as trauma, tissue necrosis, infection and myocardial infarction. The interaction between the 44-base RNA aptamer and the target analyte CRP is studied. In particular, the influence of the aptamer immobilization procedure (chemistry, length, concentration), as well as the binding conditions, i.e., the influence on the binding of different buffers, the presence of Ca2+ ion and the specificity (against human serum albumin) have been evaluated. Using the best working conditions, we achieved a detection limit of 0.005 ppm, with good selectivity towards human serum albumin. Some preliminary experiments in serum are reported.

Crocins pattern in saffron detected by UHPLC-MS/MS as marker of quality, process and traceability.

This study was carried out to develop an UHPLC-MS/MS analytical procedure, to determinate all isomers and isoforms of crocins of 42 saffron samples, with different origin, age and dried using different process conditions. A preliminary experimental design was applied to optimize the extraction of crocins; UHPLC-MS/MS conditions were set to obtain the best analytical performances in terms of sensitivity and selectivity. The optimised conditions allowed to determine ten crocins; their amount in samples was significantly different and affected by process, age and origin. Drying conditions influenced the crocins pattern and this was particularly evidenced in the more recently produced samples, with a clear separation between mild and high thermally treated samples. Principal Component Analysis of all crocins data allowed to discriminate samples based on origin (Italy vs. other countries) and age. Results confirm the feasibility of the use of crocins pattern as marker of quality and traceability of saffron.

Disposable electrochemical genosensor for the simultaneous analysis of different bacterial food contaminants.

This paper deals with the use of an electrochemical genosensor array for the rapid and simultaneous detection of different food-contaminating pathogenic bacteria. The method includes PCR amplification followed by analysis of the amplicons by hybridisation with toxin-specific oligonucleotide probes. A screen-printed array of four gold electrodes, modified using thiol-tethered oligonucleotide probes, was used. Unmodified PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes and biotinylated signaling probes. The resulting biotinylated hybrids were coupled with a streptavidin-alkaline phosphatase conjugate and then exposed to an alpha-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the alpha-naphthol oxidation signal. Mixtures of DNA samples from different bacteria were detected at the nanomolar level without any cross-interference. The selectivity of the assay was also confirmed by the analysis of PCR products unrelated to the immobilised probes.

An electrochemical bioassay for dichlorvos analysis in durum wheat samples.

The use of an acetylcholinesterase inhibition assay for the detection of dichlorvos in durum wheat samples by a simplified extraction procedure is reported. After an incubation step, the residual activity was determined with an amperometric biosensor using a portable potentiostat. The use of electric eel and recombinant acetylcholinesterase was compared. The effect of the matrix extract was evaluated by using various sample:solvent ratios, 1:2.5, 1:5, 1:10, and 1:20. The optimal extraction ratio, considering the electrochemical interferences and the effect on enzyme activity and bioavailability of the pesticide, was 1:10. Calibrations were performed in buffer and durum wheat extract. The calculated detection limits in buffer solution were 10 ng/ ml and 0.045 ng/ml for electric eel and recombinant acetylcholinesterase, respectively, whereas operating in the matrix extract they increased up to 45 ng/ml and 0.07 ng/ml, corresponding to 0.45 mg/kg (extraction ratio 1:10) and 0.07 mg/kg in samples. These characteristics allowed the detection of contaminated samples at the maximum residue limit, which is 2 mg/kg and well below. Fortified samples of durum wheat were obtained with both dichlorvos and the commercial product Didivane, which contains dichlorvos as an active molecule. At all the tested levels, the occurrence of contaminant was detected with an average recovery of 75%. The total assay time, including the extraction step, was 30 min. Because several extractions as well as most of the assay steps can be run simultaneously, the throughput for one operator is 12 determinations per hour.

Analytical applications of aptamers.

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets. Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction. Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.

A novel optical biosensor format for the detection of clinically relevant TP53 mutations.

The TP53 gene has been the subject of intense research since the realisation that inactivation of this gene is common to most cancer types. Numerous publications have linked TP53 mutations in general or at specific locations to patient prognosis and therapy response. The findings of many studies using general approaches such as immunohistochemistry or sequencing are contradictory. However, the detection of specific mutations, especially those occurring in the structurally important L2 and L3 zinc binding domains, which are the most common sites of TP53 mutations, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. In this study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments Spreeta chips were used to develop a DNA biosensor based on thiolated probes complementary to these domains. The sensors were able to detect these mutations in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but the difference in hybridisation signal was small. Preliminary experiments to enhance the signal using Escherichia coli mismatch repair proteins, MutS and single strand binding protein were carried out. It was found that MutS was unable to bind to mismatch oligonucleotides, but single strand binding protein was able to bind to single stranded probes, which had not hybridised to the target, resulting in a three-fold increase in the sensitivity of the biosensor. While further work needs to be carried out to optimise the system, these preliminary experiments indicate that the TI-SPR-1 can be used for the detection of clinically relevant mutations in the TP53 gene and that the sensitivity can be increased significantly using single strand binding protein. This system has a number of advantages over current mutation detection technologies, including lower cost, ease of sensor preparation and measurement procedures, technical simplicity and increased speed due to the lack of need for gel electrophoresis.

Aptamer-based biosensors for the detection of HIV-1 Tat protein.

Two biosensors have been constructed using an RNA aptamer as biorecognition element. The aptamer, specific for HIV-1 Tat protein, has been immobilised on the gold surface of piezoelectric quartz crystals or surface plasmon resonance (SPR) chips to develop a quartz crystal microbalance (QCM)-based and an SPR-based biosensor, respectively. Both the biosensors were modified with the same immobilisation chemistry based on the binding of a biotinylated aptamer on a layer of streptavidin. The binding between the immobilised aptamer and its specific protein has been evaluated with the two biosensors in terms of sensitivity, reproducibility and selectivity. A protein very similar to Tat, Rev protein, has been used as negative control. The two biosensors both were very reproducible in the immobilisation and the binding steps. The selectivity was high in both cases.

Characterization of monoclonal antibodies to 2,4-dichlorophenoxyacetic acid using a piezoelectric quartz crystal microbalance in solution.

Piezoelectric quartz crystals (resonance frequency 10 MHz) were used for an investigation of the immunochemical reaction between 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide and several monoclonal antibodies prepared against 2,4-D. The herbicide was immobilized on the gold electrodes of the crystals silanized with 3-aminopropyltriethoxysilane. The activated carboxylic group of 2,4-D was linked either directly to the silanized surface or through hexamethylenediamine or albumin as a macromolecule. The interaction of the immobilized antigen with monoclonal antibody in solution was followed as a change in the resonant frequency of the crystals. The best results were achieved using 2,4-D attached to albumin. The affinity binding of five monoclonal antibodies was characterized by association (ka) and dissociation (kd) kinetic rate constants and by equilibrium association constants (KA) which were obtained from the experimental frequency vs. time curves.

Development of an optical fibre sensor for ammonia, urea, urease and IgG.

An optical fibre sensor utilizing Brilliant Yellow in a thin cellulose acetate membrane as a pH sensor has been developed. By use of gas permeable membrane, urease-immobilized membrane and IgG immobilized membrane we obtained optical sensors for ammonia, urea, urease, IgG suitable for physiological and clinical applications. The system makes use of commercially available instrumentation for absorbance measurement and flow cell for fast analysis.

Sensitive detection of pesticides using amperometric sensors based on cobalt phthalocyanine-modified composite electrodes and immobilized cholinesterases.

The determination of organophosphate and carbamate pesticides was carried out using cobalt phthalocyanine-modified carbon epoxy composite electrodes coupled with acetylcholinesterase or butyrylcholinesterase. Covalent immobilization of enzymes on Immobilon membranes or nylon nets was examined; the highest sensitivity to inhibitors was found for the nylon net containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetyl- or butyrylthiocholine as substrates; thiocholine produced by hydrolysis in the enzyme membrane was electrochemically oxidized at +300 mV (vs. Ag/AgCl reference). The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, 1.5 and 8.4 micrograms l-1 of paraoxon and heptenophos, respectively, can be detected in less than 3 min. These detection limits are similar as those obtained when analyses were performed using free cholinesterase and 10 min incubation with inhibitor.

Disposable DNA electrochemical sensor for hybridization detection.

A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidinbiotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 microgram/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.

Improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals.

The high sensitivity and specificity of DNA hybridisation techniques makes them powerful tools for environmental or clinical analysis. This work describes the development of a DNA piezoelectric biosensor for the detection of the hybridisation reaction. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of oligonucleotides onto the gold surface of the quartz crystal. Four immobilisation procedures were tested and compared considering the amount of immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non-specific adsorption. All the experiments were performed with oligonucleotides of 25 bases (probe, target and non-complementary oligonucleotide). The four coating methods were all based on the use of self-assembled monolayers (SAM). Three of them employed the interaction between streptavidin and biotin for the immobilisation of a biotinylated probe. Results indicated that immobilisation of a biotinylated probe on streptavidin linked to a layer of carboxylated dextran provides higher sensitivity for the detection of the hybridisation reaction, absence of non-specific adsorption and a higher stability with respect to the regeneration step.

Surface modifications for the development of piezoimmunosensors.

Four different techniques for the immobilisation of proteins onto the gold electrode of a piezoelectric quartz crystal were investigated. The examined techniques were adsorption, avidin-biotin binding and two different types of covalent binding on self-assembled monolayers (SAM), dithiobis(succinimidylpropionate) (DSP) and a dextran modified thiol monolayer. The reaction of the immobilised proteins (bovine serum albumin (BSA) and anti-human IgG) with their specific antibodies, anti-BSA and hIgG (50 and 200 micrograms/ml) were studied using a quartz crystal microbalance and then compared. Many cycles of measurements were performed on the same crystal regenerating the gold surface with a solution of glycine.HCl, 100 mM, pH 2.1. The interactions of the immobilised reagents with non-specific antibodies were also studied. The adsorption protocol was the quickest, but did not allow regeneration with glycine.HCl. Thiol-dextran coated surfaces did not show any detectable response to non-specific reagents, but needed a very long and complicated protocol. DSP and avidin-biotin coating procedures were easy and not too long. They seemed to have the best characteristics of reproducibility among different crystals and possibility of regeneration of the coated surface, but the percentage of non-specific binding was high.

Development of biosensors with aptamers as bio-recognition element: the case of HIV-1 Tat protein.

The in vitro selection of combinatorial libraries of RNA/DNA, has allowed the identification of specific nucleic acids (aptamers) which bind to a wide range of target molecules with high affinity and specificity. In this work, an RNA aptamer, specific for the protein trans-activator of transcription (Tat) of HIV-1, has been used as bio-recognition element to develop a biosensor (aptasensor). The biosensor was optimised using piezoelectric quartz-crystals as transducers and the aptamer was immobilised on the gold electrode of the crystal. The immobilisation procedure was based on the interaction between the biotinylated aptamer and streptavidin previously deposited on the electrode. The main analytical characteristics of the biosensor, such as sensitivity, selectivity and reproducibility, have been studied in details. An optimised regeneration procedure allowed the multiple use of the aptamer-coated crystal. The aptasensor has been compared with the corresponding immunosensor, based on the specific monoclonal anti-Tat antibody. The antibody was immobilised on a layer of carboxylated dextran previously deposited on the gold electrode. The results demonstrated that the use of a biosensor with a specific aptamer as bio-recognition element could be an interesting approach in the detection of proteins, which has been here examined considering a model system.

Coupling of a DNA piezoelectric biosensor and polymerase chain reaction to detect apolipoprotein E polymorphisms.

In this paper we report the coupling of the Polymerase Chain Reaction (PCR) with a piezoelectric biosensor to detect a point mutation in a human gene. Biotinylated 23-mer probes were immobilised on the streptavidin coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridisation of the immobilised probes with a short sequence (23 mer) complementary, non-complementary and mismatched DNA was investigated: the device was able to distinguish the different synthetic oligonucleotides. Many cycles of measurements can be performed on the same crystal surface regenerating the single strand of DNA with 1 mM of HCl. The same hybridisation reaction was then performed using real samples of human DNA extracted from blood and amplified by PCR, following a standard procedure for genetic detection of the polymorphism of the apolipoprotein E (apoE) gene. The procedure was able to distinguish the sequences present in the different samples, which differ only in one base: in this way it was possible distinguish between different groups of genotypes with apoE typing. Experiments with 'blank' samples confirmed the absence of adsorption or non-specific effects on the quartz crystal treated with the reported procedure.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 1).

The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use.