Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples. I. Spectrophotometric approach to design the calibration curve for the quantification

High selectivity of a biomarker is a basic requirement when it is used for diagnosis, prognosis and treatment of a disease. The artificial gel antibodies, which we synthesise by a molecular imprinting method, have this property not only for proteins, but also for bioparticles, such as viruses and bacteria. However, diagnosis of a disease requires not only that the biomarker can be “fished out” from a body fluid with high selectivity, but also that its concentration in the sample can rapidly be determined and preferably by a simple technique. This paper deals primarily with the development of a spectrophotometric method, which is so simple and fast that it can be used with advantage in a Doctor's Office. The development of this method was not straight‐forward. However, by modifications of the performance of these measurements we can now design standard curves in the form of a straight line, when we plot the true (not the recorded “apparent” absorption) against known protein concentrations. In an additional publication (see the following paper in this issue of JSS) we show an application of such a plot: determination of the concentration of albumin in serum and cerebrospinal fluid from patients with neurological disorders to investigate whether albumin is a biomarker for these diseases.     

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria). Ib. Gel antibodies against proteins (hemoglobins)

Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.     

Highly selective chromogenic and fluorogenic probe based on benzothiazole-thiosemicarbazone Schiff base for detection of copper (II) in real samples

Copper is the third most abundant essential transition metal ion in the human body. It's responsible for important activities in many living things, but excessive intake of Cu²⁺ can lead to a range of diseases. A colorimetric and turn-off fluorescent probe (E)-2-(5-(benzothiazol-2-yl)-2-(diethylamino)-4-hydroxybenzylidene)-N-phenylhydrazine-1-carbothioamide ( ZTR ) was designed and synthesized by thiosemicarbazone Schiff base as a specific complexes site strategy to achieve highly specific Cu²⁺ detection. The fluorescence of the probe ZTR solution fell dramatically when Cu²⁺ was added, and its appearance changed from dazzling blue to nearly colorless. The simple structure and readily available fluorescent probe provide a novel approach for the quantitative detection of Cu²⁺ in the linear range from 0 to 0.12 μM, with a detection limit down to 16 nM, and with high selectivity for Cu²⁺ over 15 other metal ions. Job’s plot analysis showed that probe ZTR and Cu²⁺ formed a 1:1 coordination complex. In addition, because of its low detection limits and fast response time, the created fluorescent molecule was effectively used to study the target ions on test paper strips and in water samples.))     

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria): II. Gel antibodies against virus (Semliki Forest Virus)

Artificial and highly selective antibodies (in the form of gel granules) against proteins can easily be synthesized by a simple, cost-effective imprinting technique [Liao, J.-L. et al., Chromatographia 1996, 42, 259-262]. Using the same method for synthesis of gel antibodies against viruses in combination with analysis by free zone electrophoresis in a rotating narrow bore tube we have shown that artificial gel antibodies against Semliki Forest Virus (wild type) can sense the difference between this virus and a mutant, although they differ in their chemical composition only by three amino acids in one of the three proteins on the surface of the virus particle. The reason for this extremely high resolution is explained by the fact that we use three types of selectivity: (i) shape selectivity (created by the close fit between the antigen and its imprint in the gel), (ii) bond selectivity in the contact area between the antigen and its imprint in the gel antibody, and (iii) charge selectivity, originating from slightly different structures or/and conformations of the antigens.