Novel Biosensing Methods Based On The Cytochrome C And Nanometer Luminescence Materials

Recently, biosensors have been widely applied in clinical testing, drug screening and medical research, because they possess remarkable advantages, such as high accuracy, fast analysis speed, low-cost and good selectivity. Optical measurement technology has gained great attention in the development of biosensors, due to many advantages such as low background noise, easy operation and high sensivity. Moreover, the development of new biological materials and metal nanomaterials provide new sensing design strategies and platforms for basic research. This doctoral thesis is based on advantages of cytochrome c (Cyt c) and nanometer luminescence materials in biosensing and biochemical analysis applications, focusing on the research hotspots, developing several biosensing analysis methods for the detection of the activity of enzyme related to diseases and inhibition, protein and small molecule, respectively. Compared with the traditional approches, the developed detection methods are simple, sensitive and low-cost. The practicability of these proposed methods was also verified. The detailed contents are described as follows.The phosphorylation of cellular protein by kinases plays a pivotal regulatory role in many cellular processes. In Chapter 2, based on phosphorylation protection against CPY degradation and high quenching capacity of Cytochrome c, we develop for the first time a novel fluorescence peptide/Cyt c sensing platform for detection of casein kinase 2 (CK2) activity and inhibition. In this work, without CK2 treatment or coincubated with CK.2 inhibitor, the FITC labeled substrate peptide is promptly hydrolyzed by CPY, leading to the release of the FITC. The free FITC with reduced anionic charge would be unable to absorb on Cyt c because of their weaker electrostatic interaction between the FITC and Cyt c, and thus induce the negligible fluorescence quenching by Cyt c. In contrast, after CK2 treatment, the phosphorylated peptide S can resist CPY digestion, yielding anionic phosphopeptides in the reaction buffer. Upon the addition of Cyt c, the fluorescence was greatly quenched due to the strong absorption of peptide fragments on Cyt c by electrostatic interaction and the effective electron transfer (ET) from the FITC to the heme cofactor of Cyt c. Therefore, the activity of CK2 can be easily and facilely monitored by fluorescence signal change. It also exhibited a good assay performance in cell lysates and satisfactory recoveries were obtained. Moreover, our constructed method can be applied to the detection of cyclin-dependent kinase (CDK1) activity.In Chapter 3, based on the strong affinity of aptamer and lysozyme, a new, label free aptamer fluorescent biosensor was successfully developed for lysozyme assay. At first, aptamer functionalized CdTe QDs (DNA-CdTe QDs), which served as fluorescent probe, was directly synthesized by a one-pot method. The negatively charged DNA-CdTe QDs can bound quickly on cationic Cyt c through electrostatic interaction under our experimental conditions. The fluorescence of QDs was significantly quenched due to the the effective ET from the QDs to the heme cofactor of Cyt c. In the presence of lysozyme, the specific binding between the aptamer and lysozyme caused dissociation of the DNA-CdTe QDs/Cyt c complex, resulting in the disruption of ET process. A fluorescence restoration could be observed. Therefore, the fluorescence intensity changes were directly related to the amount of the lysozyme added in the reaction solution. This approach did not involve fluorescent labeling on aptamer probes, making it costless. In addition, a high sensitivity and selectivity was also obtained due to the high quenching capacity of Cyt c. Moreover, this proposed method can readily expand to detect other protein by displacing the aptamer sequence.β-secretase (BACE1) is a key enzyme responsible for generation of neurotoxic amyloid peptide and regarded as an important target in the treatment of Alzheimer’s disease (AD). In Chapter 4, a new label-free fluorescence method was proposed for sensitive and selective detection of BACE1 as well as the inhibitors, based on growth of fluorescent CdS quantum dots(QDs) by enzymatic products. Herein, a dithiol peptide probe was designed as a BACE1 substrate peptide. The hydrolysis process was initiated by BACE1 between leucine and aspartic acid to produce two monothiol peptides, which triggers the formation of CdS QDs and generates a fluorescent signal. In contrast, without BACE1 treatment or coincubation with the BACE1 inhibitor, uncleaved dithiol peptide would inhibit the QDs growth, because dithiol substrate peptide have a much higher affinity with crystal surface owing to the bidentate chelate effect. Compared with the traditional FRET-based approaches, the proposed strategy is simple and low-cost without any dye labels or complicated probe design.Diabetes mellitus is a common metabolic disease, it is of utmost importance to frequently monitor and tightly control the change of blood sugar concentration for effective diagnosis and management of diabetes. In Chapter 5, we reported a novel upconverting hybrid nanocomposite, composed by DNA-templated AgNPs (DNA-AgNPs) and NaYF4:Yb/Tm@NaYF4 core-shell upconversion nanoparti- cles (UCNPs), for H2O2 and glucose sensing. This nanocomposite is based on luminescence resonance energy transfer (LRET) between UCNPs with bared surface as donor and DNA-AgNPs as efficient quenchers. In this study, we connected a cytosine-rich sequence with a poly(adenine) sequence and used it as the scaffold to synthesize AgNPs through a one-step process. DNA-AgNPs is revealed to directly assemble on the bared surface of UCNPs for the first time in this work. The LRET process was initiated by coordination interaction between the phosphonate groups of DNA-AgNPs and the exposed lanthanide ions of UNCPs, which thereby quenched the upconversion luminescence (UCL) of donor. However, AgNPs could be etched by H2O2 and transformed to Ag+, and the energy transfer was thus blocked, causing the recovery of UCL. On the basis of the conversion of glucose into H2O2 by glucose oxidase, the DNA-AgNPs/UCNP nanocomposite was further exploited for detecting glucose in blood. This method require the assistance of costly other enzyme, which makes the analysis costless. The method showed good sensitivity and the limit of detection for glucose and H2O2 was 1.08 μM and 1.41 μM, respectively. Meanwhile, it worked well in complex biosamples.Determination of alkaline phosphatase (ALP) activity has important guiding significance in the clinical diagnosis of related diseases. In Chapter 6, we used single strand DNA (ssDNA)-templated Ag nanocluster (DNA-AgNCs) as a fluorescent indicator.On the basis of the fluorescence enhancement effect when DNA-AgNCs are in proximity to guanine-rich DNA sequences, and ALP could protect against λ exonuclease (λ exo) activity by a dephosphorylating DNA, a new, label free fluorescent method for detection of ALP activity was developed for the first time. In this work, we designed two DNA sequences, phosphorylated G-DNA serves as the substrate of ALP. A-DNA contained a sequence for preparation of AgNCs and a sequence complementary to the hybridization part of the G-DNA. G-DNA is promptly degraded by λ, exonuclease (λ exo) into the mono-oligonucleotides. Because of the absence of G-DNA in proximity to AgNCs, a low fluorescent readout was obtained. However, if ALP is introduced into the system, the phosphorylated G-DNA was hydrolyzed by ALP, and the resulting hydroxyl end significantly reduces the ability of λ exo. Because the G-DNA can hybridize with DNA-AgNCs, resulting in the guanine-rich DNA sequences close to the Ag NCs and a strong fluorescence enhancement. Therefore, ALP activity could be successfully quantified by monitoring the changes of fluorescence singal. Besides, this method can be applied for inhibitor screening of ALP, and it also exhibited a good assay performance in complex sample.