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A binding assay utilizes the highly specific molecular recognition properties of a biological macromolecule, e.g. an antibody, receptor or DNA/RNA probe for the detection/quantification of a target analyte in a complex mixture. The recognition event is linked to a signal-producing system. Because biomolecules can be easily labeled with biotin, the biotin–streptavidin interaction has been established as a general system for linking molecular recognition with signal generation. To this end, we report a rapid and simple method for conjugation of the highly detectable recombinant photoprotein aequorin with streptavidin, thus generating a universal reagent for binding assays. The method is based on the use of aequorin fused to a hexahistidine tag at the amino terminus. Thiol groups were introduced to aequorin whereas streptavidin was derivatized with maleimide groups. The conjugate was purified in a single step by immobilized metal ion affinity chromatography, thus avoiding laborious chromatographic procedures. The performance of aequorin–streptavidin conjugate was tested in a DNA hybridization assay as a model. The limit of detection was 0.3 pmol l −1 and the analytical range extended up to 500 pmol l −1 . The CV was about 8%. Besides the high detectability, the assay is rapid (completed in just 25 min) due to the flash-type (3 s) bioluminescent reaction of aequorin, which avoids substrate incubation steps that are common to binding assays employing enzyme labels.

Combined screening of the mRNAs for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) has been proposed as a more useful test than the separate PSA mRNA or PSMA mRNA assays in the molecular diagnosis and monitoring of prostate cancer. We have developed a simple and highly sensitive method for the simultaneous detection of PSA mRNA and PSMA mRNA using duplex RT-PCR and a chemiluminometric hybridization assay. Total RNA from peripheral blood was reverse-transcribed using oligo(dT)20 primer followed by duplex PCR in the presence of two pairs of primers specific for PSA and PSMA. Heat-denatured biotinylated PCR products were hybridized with PSA- and PSMA-specific oligonucleotide probes immobilized in microtiter wells. The hybrids were determined by using a streptavidin–alkaline phosphatase conjugate and a chemiluminogenic substrate. Using the duplex PCR, 50 copies of PSA DNA and 5 copies of PSMA DNA can be detected with a signal/background ratio of 9.7 and 22, respectively. Analysis of samples containing total RNA corresponding to 0.04–400 LNCaP cells indicated that the detectability of the proposed method is 1 cancer cell equivalent in 10 ml of peripheral blood. The overall reproducibility of the duplex assay, including reverse transcription, PCR, and hybridization assay, ranged from 3.6 to 15.5%. The method is very simple, rapid, sensitive and suitable for high-throughput screening analysis as opposed to the commonly used nested RT-PCR assays with electrophoretic detection.

Terbium sensitized fluorescence was used as a post-column detection system to develop a simple, sensitive and rapid high-performance liquid chromatographic method for the simultaneous determination of catecholamines norepinephrine (NE), epinephrine (E) and dopamine (DA). Catecholamines were separated by an ion-pair reversed-phase chromatography on a BDS-Hypersil analytical column with a mobile phase of methanol and 50 mmol l −1 acetate buffer (pH 4.7) containing 1.1 mmol l −1 SOS and 0.11 mmol l −1 EDTA (15+85 v/v). Catecholamines and the internal standard (3,4-dihydroxybenzylamine, DHBA) were post-column derivatized by the addition to the eluent of an alkaline solution containing a stoichiometric mixture of terbium(III) chloride and EDTA. Fluorescence detection ( λ ex =300 nm, λ em =545 nm) is based on the sensitization of terbium ion fluorescence after complexation with catecholamines. The chemical compatibility between the eluent and the post-column reagent was studied and the analytical characteristics of the method were established. Detection limits found were 1.0×10 −8 , 4.0×10 −8 and 7.0×10 −8 mol l −1 for NE, E and DA, respectively. The method has been successfully applied to the determination of catecholamines in urine samples after solid-phase extraction (SPE) pre-treatment. Recoveries from urine spiked with NE (4.0×10 −7 , 2.0×10 −6 and 4.0×10 −6 mol l −1 ), E (8.2×10 −8 , 4.1×10 −7 and 8.2×10 −7 mol l −1 ) and DA (1.0×10 −6 , 5.0×10 −6 and 1.0×10 −5 mol l −1 ) varied from 98 to 100% (mean=99.3%), from 106 to 107% (mean=106.3%) and from 98 to 101% (mean=99.3%), respectively. The between-run precision (relative standard deviation, R.S.D.) for the method for three urine samples at different concentration levels of each catecholamine varied from 3.6 to 7.0%.

There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp. The 'diagnostic' membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3-8.2.

For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896 bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ~2 h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood.