Novel Biosensing Technologies Of Functional Nanomaterials And Their Applications In Disease Theranostics

Nanotechnology,developed in the 1990s,is a new hotspot technology,it showed great potential applications cross field especially in chemistry and biology.As the core of nanotechnology,functional nanomaterials,due to their unique electrical,chemical,magnetic and mechanical properties,provide a simple way for complex biological research and solve many problems that are difficult to achieve with traditional biological methods.By applying nanomaterials to biosensors,a kind of nanobiosensor has been formed,which involves interdisciplinary fields of biology,chemistry,nanoscience and so on.It has greatly promoted the development of various new biosensors with different detection and sensing mechanism and their applications in the diagnosis and treatment of diseases.Meanwhile,the emergence of new biosensors with good selectivity,high sensitivity,rapid response,low detection cost and easy operation has in turn promoted the rapid development of various disciplines such as biomedic ine,biochemistry,nanoscience and bioscience.This doctoral thesis,based on the functional nanomaterials and functional nucleic acid amplification strategy,developed several new biosensing methods for the detection of phosphatase activity,telomerase activity in different cells,microRNA in cells and in vivo,and the study of gene editing within living cells.It has also realized the sensing detection of cancer and other major diseases,imaging diagnosis in living cells and in vivo,and disease therapy by gene editing technology.The detailed research contents are described as follows:Telomerase,a specific reverse transcriptase,can elongate repetitive sequences?TTAGGG?to the end of telomeres by using its template strand primer?TS primer?.To date,it is well-known that telomerase plays an active role in mediating various physiological processes including cell growth,senescence,as well as carc inogenesis.Usually,in normal cells,the telomerase activity is repressed or absent;however,in almost all cancer cells,telomerase is overexpressed,and it is thought to be responsible for the continuous and uncontrolled growth of cancer cells.Therefore,telomerase has become one of important and credible biomarkers for cancers,and so,the methods for detection of telomerase activity are very crucial not only for early cancer diagnostics but also for better understanding of disease mechanisms.In chapter 2,we developed a novel multivalent self-assembled DNA polymer,constructed through telomerase primer sequence(I_TS)triggered hybridization chain assembly using two functional hairpin probes?tumor-trageting aptamer modified H1 and signal probe modified H2?,for sensitive detection and imaging of telomerase activity in living cells.After internalizing into the tumor cells by multivalent aptamer targeting,the I_TS on DNA polymers can be elongated by intracellular telomerase to generate telomere repeat sequences that are complementary with the signal probe,which can proceed along the DNA polymers,and gradually light up the whole DNA polymers,leading to an enhanced fluorescence signal directly correlated with the activity of telomerase.Our results demonstrated that the developed DNA polymer show excellent performance for specifically detecting telomerase activity in cancer cells,dynamically monitoring the activity change of telomerase in response to telomerase-based drugs,and efficiently distinguishing cancer cells from normal cells.The proposed strategy may afford a valuable tool for the monitoring of telomerase activity in living cells and have great implications for biological and diagnostic applications.Molecular bioimaging relative expression levels of cancer-related biomarker in vivo is rapidly emerging as a powerful strategy for accurate disease diagnosti cs.The development of noninvasive imaging probes for the determination of specific biomarker in vivo is critically required for cancer diagnosis.However,during the onset and progression of cancer,the dynamic living system complexity makes it difficult to track and visualize in vivo cancer-related biomarker,especially biomarker with low expression level,owing to their inadequate signal output.We can convert various targets including RNA,proteins,and small molecule biomarkers detection into detection of amplifiable DNA,so as to realize the sensitivity detection of the response signal.In chapter 3,we designed a novel nanoplatform to realize catalytic hybridization cascades reaction on a localized self-assembled DNA polymer for fast and highly sensitive in vivo tumor-targeting microRNA imaging.It was confirmed that under conditions of restricted and compact space?that is,limited diffusio n?,cascades interactions between DNA probes are effectively accelerated,facilitating the reduced time and enhanced sensitivity.When applied to in vivo tumor-targeting microRNA imaging,an amplified fluorescence signals can be fast achieved in the MCF-7 tumor sites in mice.Compared to the conventional catalytic hairpin assembly?CHA?strategy,our method could substantially reduce the response time and enhanced image contrast.The design concept can be widely adapted to other cancer cell-specific DNA probes for in vivo molecular imaging of cancer.The CRISPR/Cas9,clustered regularly interspaced short palindromic repeat?CRISPR?and CRISPR-associated proteins 9?Cas9?,is a promising genome editing technology that offers revolutionary solutions to genetic diseases.CRISPR/Cas9-based gene editing typically uses engineered single guide RNA?sgRNA?for targeting double-stranded DNA sequence with a protospacer adjacent motif?PAM,a short DNA sequence of the form 5'-NGG-3',where“N”=any nucleotide?,and the subsequent introduction of a double-strand break on the genome by the Cas9 protein,so as to achieve the effect of gene editing.In chapter 4,we report the first example of DNAzyme-controlled editing system,by conjugating Cas9/sgRNA loaded proteins caffolded nanoassembly and DNAzyme,for regulable transport of both Cas9 protein and sgRNA into cellnucleus for gene editing.It was shown that SA-scaffolded DNA nanoassembly efficiently deliver Cas9 protein and sgRNA to the cytoplasm via endocytosis pathway followed by endosomal escape,with concomitant DNAzyme-controlled release and transport to the nucleus,leading to high efficient genome editing in live cells.This work may offer a new avenue for exceptionally improved genetic materials delivery by precisely engineered biocompatible SAscaffolded CRISPR-Cas9 nanoassembly.Inorganic pyrophosphatase?PPase?is a ubiquitous enzyme which plays an vital role in controlling the level of PPi in cells.PPase has been proved to be relevant to evolutionary events,phosphorus metabolism and carbohydrate metabolism,which is of vital importance for cell growth and differentiation.Furthermore,the level of PPase is connected to several diseases,such as hyperthyroidism,lung adenocarcinomas and colorectal cancer.Thus,the analysis of PPase activity is of fundamental importance and significance.In chapter 5,we report the development of a novel fluorometric method for highly sensitive detection of PPase using the GQD nanoprobe.This method relies on our new finding that carboxyl-modified GQDs can be assembled by coordination with Cu²⁺into large aggregates,which can be reversely disaggregated into GQDs by the addition of PPi.The reversible modulation of fluorescence signal by Cu²⁺and PPi in the aggregation and disaggregation enables sensitive detection of PPase.In the presence of Cu²⁺,GQDs are assembled into aggregates due to the crosslinked coordination between Cu²⁺and carboxyl group,which results in substantial fluorescence quenching because of self-quenching between GQDs.After the addition of PPi,the competition of PPi with GODs for Cu²⁺due to strong interaction between PPi and Cu²⁺induces disaggregation of GODs,recovering the fluorescence of GODs.The presence of active PPase catalyzes the hydrolysis of PPi into phosphate,which releases Cu²⁺and,in turn,mediates re-aggregation of GQDs with a quenched fluorescence signal.Therefore,the modulation of fluorescence in response to PPase gives a quantitative measure for the assay of activity or inhibition of PPase.