Using force spectroscopy analysis to improve the properties of the hairpin probe

The sensitivity of hairpin-probe-based fluorescence resonance energy transfer (FRET) analysis was sequence-dependent in detecting single base mismatches with different positions and identities. In this paper, the relationship between the sequence-dependent effect and the discrimination sensitivity of a single base mismatch was systematically investigated by fluorescence analysis and force spectroscopy analysis. The same hairpin probe was used. The uneven fluorescence analysis sensitivity was obviously influenced by the guanine-cytosine (GC) contents as well as the location of the mismatched base. However, we found that force spectroscopy analysis distinguished itself, displaying a high and even sensitivity in detecting differently mismatched targets. This could therefore be an alternative and novel way to minimize the sequence-dependent effect of the hairpin probe. The advantage offered by force spectroscopy analysis could mainly be attributed to the percentage of rupture force reduction, which could be directly and dramatically influenced by the percentage of secondary structure disruption contributed by each mismatched base pair, regardless of its location and identity. This yes-or-no detection mechanism should both contribute to a comprehensive understanding of the sensitivity source of different mutation analyses and extend the application range of hairpin probes

Sensitive Fluorescence Assay for the Detection of Alkaline Phosphatase Based on a Cu2+-Thiamine System

The authors describe a novel, facile, and sensitive fluorometric strategy based on a Cu2+-thiamine (Cu2+-TH) system for the detection of alkaline phosphatase (ALP) activity and inhibition. The principle of the method is as follows. Under a basic conditions, TH, which does not exhibit a fluorescence signal, is oxidized into fluorescent thiochrome (TC) by Cu2+. Ascorbic acid 2-phosphate (AAP), which is the enzyme substrate, is hydrolyzed to produce ascorbic acid (AA) by ALP. The newly formed AA then reduces Cu2+ to Cu+, which prevents the oxidation of TH by Cu2+; as a result, the fluorescent signal becomes weaker. On the contrary, in the absence of ALP, AAP cannot reduce Cu2+; additions of Cu2+ and TH result in a dramatic increase of the fluorescent signal. The sensing strategy displays brilliant sensitivity with a detection limit of 0.08 U/L, and the detection is linear in the concentration range of 0.1 to 100 U/L. This approach was successfully applied to ALP activity in human serum samples, indicating that it is reliable and may be applied to the clinical diagnosis of ALP-related diseases.</jats:p

A label-free fluorescence method for actin detection based on DNA-templated silver nanoclusters

A facile fluorescence method has been developed for the detection of actin based on an enzyme-responsive DNA-silver nanocluster probe.</p

Fluorescent Method for the Detection of Biothiols Using an Ag+-Mediated Conformational Switch

In this work, a novel, simple, and time-saving fluorescence approach for the detection of biothiols (glutathione and cysteine) was developed by employing a DNA probe labeled with 2-aminopurine. As an adenine analogue, 2-aminopurine exhibits high fluorescence intensity that can be rapidly quenched in the presence of DNA. In the presence of Ag+, the fluorescence increased significantly, which was a result of the formation of cytosine&ndash;Ag+&ndash;cytosine base pairs and the release of 2-aminopurine. Upon addition of either glutathione or cysteine, the structure of cytosine&ndash;Ag+&ndash;cytosine was disrupted, a product of the stronger affinity between biothiols and Ag+. As a result, the 2-aminopurine-labeled DNA probe returned to its former structure, and the fluorescence signal was quenched accordingly. The detection limit for glutathione and cysteine was 3 nM and 5 nM, respectively. Furthermore, the determination of biothiols in human blood serum provided a potential application for the probe as a diagnostic tool in clinical practice

Functional Nucleic Acid Probes Based on Two-Photon for Biosensing

Functional nucleic acid (FNA) probes have been widely used in environmental monitoring, food analysis, clinical diagnosis, and biological imaging because of their easy synthesis, functional modification, flexible design, and stable properties. However, most FNA probes are designed based on one-photon (OP) in the ultraviolet or visible regions, and the effectiveness of these OP-based FNA probes may be hindered by certain factors, such as their potential for photodamage and limited light tissue penetration. Two-photon (TP) is characterized by the nonlinear absorption of two relatively low-energy photons of near-infrared (NIR) light with the resulting emission of high-energy ultraviolet or visible light. TP-based FNA probes have excellent properties, including lower tissue self-absorption and autofluorescence, reduced photodamage and photobleaching, and higher spatial resolution, making them more advantageous than the conventional OP-based FNA probes in biomedical sensing. In this review, we summarize the recent advances of TP-excited and -activated FNA probes and detail their applications in biomolecular detection. In addition, we also share our views on the highlights and limitations of TP-based FNA probes. The ultimate goal is to provide design approaches for the development of high-performance TP-based FNA probes, thereby promoting their biological applications

Entrapment of Individual DNA Molecules and Nanoparticles in Porous Alumina Membranes

Depth-resolved fluorescence imaging allows the motion of single DNA molecules and single nanoparticles at the liquid/solid interface to be recorded in real time. Porous alumina membranes were employed as model chromatographic packing material. Using a suitable pH and ionic strength, adsorptive interactions are suppressed. The effects of 3-dimensional topography, specifically the presence of nanopores, on DNA and nanoparticle migration across the surface are, thus, revealed. The residence times and the number of immobilized DNA molecules or particles increased as the pores size increased. Yet, we found that the pore diameter must be significantly larger than the particle diameter or the DNA short radius before entrapment can occur. Furthermore, the depth distribution of particles does not conform to one-dimensional diffusion in the pores, probably because of collisions with the walls. These observations provide new insights into conventional liquid chromatography as well as size-exclusion chromatography and membrane separations