The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate-coelenterazine (CTZ), catalyzed by luciferase. Despite the ...diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization-Ca
-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme-substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.
NanoLuc (NLuc) is an artificial coelenterazine-dependent luciferase generated from the deep-sea shrimp Oplophorus gracilirostris. Its peculiar propertiessmall size and long-lasting bright ...bioluminescence triggered with the synthetic substrate furimazinehave made this enzyme popular as a reporter in a variety of analytical systems. Basically, to ensure the assay specificity, NLuc is genetically fused to the polypeptide with affinity for the corresponding target. The approach, however, has a limitation for non-protein biospecific molecules, thus forcing the production of biospecific luciferase derivatives via chemical conjugation. Unfortunately, it yields a heterogeneous product and often results in the loss of a significant part of bioluminescence activity. Here, we report on NLuc site-directed conjugation by combining these two approaches: several luciferase derivatives, genetically extended with hexapeptides carrying a unique Cys residue, were obtained, and the variant with activity equal to that of the intact NLuc was found. Biospecific molecules of the most commonly used types (low-weight hapten, oligonucleotide, antibody, and DNA aptamer) were chemically attached to this NLuc variant through the unique Cys using an orthogonal conjugation approach. The resulting conjugates were tested as labels in the bioluminescence assay and were shown to detect the corresponding molecular targets (e.g., cardiac markers) with high sensitivity.
•We patterned single-crystal graphene sheet into graphene field-effect transistor (G-FET) biosensor.•Using G-FET biosensor, we measured binding kinetics and affinity between drug imatinib and its ...target protein.•The sensor can specifically recognize the imatinib with detection limit down to 15.5 fM.•The sensor showed a linear current response to the logarithm of imatinib concentrations from 0.1 pM to 10 μM.•We studied Abl1 mutation and Mg2+ dependency of Abl1 binding to imatinib by the G-FET sensor.
We synthesized large-area single-crystal graphene sheets to use them in biosensors based on field-effect transistors (FET) for quantitative analysis of interaction kinetics and affinity between the imatinib drug and its target protein kinase Abl1. The G-FET biosensor showed an excellent performance and recognized imatinib at as low as 15.5 fM. The biosensor also showed a linear response to the logarithm of imatinib concentration in the 0.1 pM-10 μM range. This graphene-based FET biosensor (G-FET) was also applied to quantify Abl1 Y253 F mutation and Abl1 dependency on Mg2+ to bind to imatinib in real-time. Results demonstrated in this work clearly showed that the novel G-FET biosensors are very promising to analyze interactions between proteins and low molecular weight drugs.
Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and ...apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering “character”.
Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic ...characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins (cardiac troponin I, survivin, and melanoma inhibitory activity protein) bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100-500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated.
Bioluminescent solid-phase sandwich-type microassay was developed to detect multiple sclerosis (MS)-associated autoantibodies in human sera. The assay is based on two different 2′-F-Py RNA aptamers ...against the target autoantibodies as biospecific elements, and Ca2+-regulated photoprotein obelin as a reporter. The paper describes elaboration of the assay and its application to 91 serum samples from patients with clinically definite MS and 86 ones from individuals healthy in terms of MS. Based on the receiver-operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers sensitivity of 63.7% and specificity of 94.2%. The area under the ROC curve (AUC) value of 0.87 shows a good difference between the groups under investigation. The likelihood ratio of 10.97 proves the diagnostic value of the assay and its potential as one of the laboratory MS-tests.
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•Assay of multiple sclerosis-associated autoantibodies was developed using RNA aptamers and photoprotein obelin.•This bioluminescent assay was successfully applied to analyze 177 clinical sera samples from MS patients and healthy donors.•Its diagnostic value and potential as one of the laboratory tests for multiple sclerosis diagnosis were demonstrated.
Bioluminescent solid‐phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer–target complexes during design and development of the ...specific aptamers. The assay involves Ca2+‐regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.
Ca2+‐regulated photoprotein obelin was shown to be an excellent reporter in the solid‐phase assay to determine the binding characteristics of aptamer–target complexes in the design and development of highly specific DNA aptamers.
A top-down nanofabrication approach involving molecular beam epitaxy and electron beam lithography was used to obtain silicon nanowire-based back gate field-effect transistors with Schottky contacts ...on silicon-on-insulator (SOI) wafers. The resulting device is applied in biomolecular detection based on the changes in the drain-source current (IDS). In this context, we have explained the physical mechanisms of charge carrier transport in the nanowire using energy band diagrams and numerical 2D simulations in TCAD. The results of the experiment and numerical modeling matched well and may be used to develop novel types of nanowire-based biosensors.
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•A simple approach for fabrication of a silicon nanowire field-effect transistor for protein detection.•An evaluation of the biosensor performance with model molecules.•An explanation of electrical detection mechanism.•A demonstration of the energy bands shifting.•2D simulation considering the biosensor surface charge concentration.
Two high-affinity DNA aptamers for lung tumor cells were applied as biospecific elements in bioluminescent assay of patient blood. The oligonucleotide complementary to the 5′ end of both aptamers ...carrying either biotin or Ca2+-regulated photoprotein obelin was used to form a sandwich-type analytical complex on the surfaces of magnetic streptavidin-activated microspherical particles. Clinical blood samples from cases of morphologically confirmed lung cancer and control samples were analyzed applying the developed assay. From the receiver operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers the sensitivity of 91.5% and the specificity of 75% (p < 0.001). The area under ROC curve with the value of 0.901 distinguishes well between the two groups under investigation.
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•A couple of DNA aptamers formed specific sandwich-type complex with tumor cells targets circulating in the bloodstream.•Magnetic microparticles with covalently immobilized streptavidin provides the complex immobilization.•Ca2+ -regulated photoprotein obelin serves as a high-sensitive bioluminescent reporter.•The assay was tested on the model and significant amount of clinical blood samples and demonstrated high sensitivity of 91.5% and the specificity of 75%.
Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer-target complexes during design and development of the ...specific aptamers. The assay involves Ca
-regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.
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