| Colorectal cancer(CRC)is a kind of common gastrointestinal cancer with an increasing incident rate in recent years.Robust screening methods like fecal occult blood test and colonoscopy have improved the diagnosis rate of colorectal cancer.However,non-invasive blood-based screening approaches are still gaining increased attention for the early detection and attenuation of mortality associated with CRC.The majority of current screening approaches are inadequate at replacing the conventional CRC diagnostic procedures(Rectal examination,imaging examination,colonoscopy,pathological examination).Yet,carcinoembryonic antigen(CEA),as a diagnostic marker,is of little value in the diagnosis of early stage of CRC.With technological advances and further research into cancer related molecules,exosomes are considered a potential new biomarker.Exosome is a kind of microvesicle with a diameter of 30-200 nm.Almost all the cells release exosomes into peripheral blood,urine,saliva,tissue fluid and other biofluids through the process of endoplasmic reticulum transmission and exocytosis.Exosomes contain nucleic acids and proteins that reflect and typify host-cell molecular architecture.Intriguingly,exosomes released from cancer cells have a distinct genetic and epigenetic makeup,which allows them to undertake their signal transmission and tumor metastasis function.In the process of secreting exosomes,the tumor cells of CRC assemble specific molecules on the membrane as well as in the vesicles,such as A33,Ep CAM,CEA.From a clinical standpoint,these unique cancer-specific molecules present in exosomes appear to be detectable in a small amount of blood,making them very attractive substrates for developing cancer biomarkers,particularly noninvasive diagnostic approaches.However,exsome's nanoscale and the low concentration occurring in the early stage of disease make them hard to be analyzed by current methods.Therefore,sensitive and rapid detection of exosomes remains challenging and urgent.I.The application of new nanomaterials in trace detection is more and more extensive.Graphene oxide(GO)has been widely used in biomolecules and living cells detection due to its good biocompatibility and large specific surface area.Aptamer,single-stranded oligonucleotide,is able to specifically detect targeted analytes(including small molecule compounds,proteins,living cells and exosomes,etc.).The binding between aptamers and their targets is of high affinity and specificity.GO can be combined with aptamers through intermolecular forces,causing changes of chemical characteristics.Several studies have been conducted to detect small molecular compounds and living cells using the interaction between GO and aptamers.However,there are no reports on using GO and fluorescence DNA single-stranded aptamers to detect exosomes.In the first part of this paper,a rapid fluorescence detection method based on the interaction between GO and fluorescence DNA single-strand aptamers(targeted on CD63 and Ep CAM)for CRC exosomes was established.The system utilized Fluorescent-aptamer-GO complexes as probes to undergo conformational change upon interaction with the CRC exosomes,resulting in fluorescence changes due to the fluorescence quenching and recovering properties of GO by adsorption and desorption of Fluorescent-aptamers.During the experiment,we optimized the reaction conditions,and aided deoxyribonuclease to amplify the fluorescence signal.The LOD of this method to detect CRC exosomes was 2.1×104 particles/?l,and the test could be done in 30 minites.The method was verified with 19 cases of clinical serum samples(5 ?l)and showed effective ability to distinguish healthy control and CRC patients(P < 0.05).This method has potential application value in clinical laboratory testing.However,the target of this detection system was Ep CAM,which was also overexpressed in pancreatic cancer and breast cancer.Therefore,the specificity of this method needs to be further improved.In the next step,the specificity and sensitivity of CRC exosomes detection will be mainly studied.II.Mass spectrometry has the characteristics of high sensitivity,wide dynamic range and high specificity.It has been widely used in the field of disease diagnosis,microbiological detection and drug analysis.Some new ambient ionization techniques have been developed in order to simplify the method of mass spectrometry,especially paper spray(PS)ionization.PS is efficient,simple and no need of pretreatment of samples.However,PS shows poor sensitivity when detecting analyte from bio-samples,because no pretreatment procedures are carried out and the matrix of bio-sample is complex with various molecules(ions,proteins).Protein detection data obtained by mass spectrometry is hard to analyze due the large molecular weight.If biomolecules are labled with cleavable small molecule compounds,the detection efficiency of quantitative analysis of biological macromolecules can be greatly improved by directly detecting small molecular compounds.To further improve the detection sensitivity of CRC exosomes,a method PS mass spectrometry based on immunoassay was established in the second part of this paper.We fabricated a double-layer paper chip to capture CRC exosomes with specific antibody(anti-CD63 antibady),and two ion probes with small MW were constructed.The CRC exosome detective antibody(anti-A33 antibody)was labled with ion probes,followed by cleaving the probe and MS analysis.In this part,the integrated analysis of immunoassay isolation and MS for small molecular ion probes was realized by using paper chip.The LOD of this method using two kind of ion probes to detect CRC exosomes were 1.7×102 particles/?l and 2.5×102 particles/?l,respectively.The sensitivity of this method was 100 times than the GO method in the first part.Furthermore,after immune capture of CRC exsomes,the paper chips showed that the reaction system was tolerant to long storage times(30 days).By using the proposed MS immunoassay protocol,the assay can be interrupted,stored,and restored and can be used for self-service for the patient.In this paper,two kinds of rapid detection platforms for CRC exosomes were developed by using novel nanomaterials and paper spray ionization mass spectrometry,respectively.Both methods were time-saving,easy to operate and with high sensitivity,and have a great application potency in the clinical diagnosis of cancer exosomes. |
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