| The integration of signal reporter and molecular recognition is the basis for the target detection and biological labeling and imaging. Since the discovery of aptamer in1990s, they have been developed as a new recognition molecule during the past thirty years. Their applications in various subjects have attracted extensive attention from researchers, such as chemistry, biomedicine, nanomaterials and physics. The combination of aptamers with novel nanomaterials such as metal clusters、magnetic nanoparticle and so on, including nanomaterial-based aptamer bioconjugates has attracted considerable interest and has led to a wide variery of applications. Fluorescent nanomaterials can be used as signal reporter due to their excellent fluorescence properties. On the other hand, MALDI-TOF MS is an useful detection method in the life anlysis field with their fast、simple、high sensitivity and good accuracy advantages.Combining the specific recognition ability of aptamer and excellent physicochemical properties of nanomaterials, we designed and constructed new types of protein sensors with optical and mass spectra as detection signals. The major contents were described as follows.1. Binding-Induced Fluorescence Turn-on Assay Using Aptamer-Functionalized Silver Nanocluster DNA ProbesWe presented a binding-induced fluorescence turn-on assay for human a-thrombin detection. Key features of this assay include affinity binding-induced DNA hybridization and fluorescence enhancement of silver nanoclusters (AgNCs) using guanine-rich DNA sequences.Two aptamers (Apt15and Apt29) were used and were modified by including additional sequence elements. A12-n.t. sequence was used to link the first aptamer with a nanocluster nucleation sequence at the5’end. The second aptamer was linked through a complementary sequence (12-n.t.) to a G-rich overhang at the3’end. Binding of the two aptamer probes to the target protein initiates hybridization between the complementary linker sequences attached to each aptamer, and thereby bring the end of G-rich overhang to close proximity to AgNCs, resulting in a significant fluorescence enhancement. This fluorescence assay is performed in a single tube, and it does not require washing or separation steps. The principle of the binding-induced DNA hybridization and fluorescence enhancement of Ag NCs can be extended to other homogenous assay applications.2. Aptamer-Magnetic Nanoparticle Conjugate-Based MALDI-TOF MS for Multiple Proteins DetectionWe described an aptamer-magnetic nanoparticle conjugate-based MALDI-TOF MS platform for multiple proteins recognition、seperatio、enrichment and detection, coupling the highly specific recognition capabilities of aptamers to the corresponding target and the great separation ability of Fe3O4by the application of a magnetic field. Aptamers were linked to the surface of Fe3O4@AuNPs through Au-S bond and used to capture the proteins. In a magnetic field, the proteins can be separated and enriched from the solution. Then, we add peptide SR-6as the internal standard and use MALDI-TOF MS to detect the tryptic digests of proteins. Human α-thrombin and platelet derived growth factor (PDGF-BB) were chosen as model proteins. We first used double signal peptides to identify and quantify thrombin and extended to detect PDGF-BB, demonstrating the good versatility of the assay. The introduction of aptamer not only assured the high efficiency of specific recognition but also brought the assay with good versatility, not producing mass spectrum peak to disturb the detection of targets. Meanwhile, by the use of MALDI-TOF MS to detect the tryptic digests, the resulting mass signal intensity was stronger, improving the detection sensitivity and lowering the detection limit. Futhermore, this work showed the potential of apt-magnetic nanoparticle conjugate-based MALDI-TOF MS in protein detection, even in the more complex, protein-rich matrices and would open the possibility for uses in enzymolysis identification of proteome. |
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