Research And Application Of Biochip-mass Spectrometry Techniques

Biomass spectrometry is one of the commonly used methods for life analysis.It has the advantages of high sensitivity and no labeling,and can provide basic structure and quantitative information of specific analytes.Matrix-assisted laser desorption ionization(MALDI)and electrospray ionization(ESI)were two "soft ionization"techniques developed in the late 1980s,and their development has increased the range and sensitivity of mass spectrometry and promoted the development of mass spectrometry in the field of life chemistry.Biochip-mass spectrometry technology,as a fourth-generation mass spectrometry platform after gas chromatography-mass spectrometry,liquid chromatography-mass spectrometry and capillary electrophoresismass spectrometry,has been widely used in the analysis of complex biological samples.Biochips play an important role in pre-processing steps such as sample preparation,sample enrichment,etc.prior to mass spectrometry,and have the potential for high throughput,automated,and rapid analysis.This dissertation combines mass spectrometry platform with technologies of nanomaterial matrix,immunoaffinity enrichment,array chip and microfluidic chip to develop new chip-mass spectrometry platforms and develop a series of rapid qualitative and quantitative methods for small molecule drugs.It contains the following five parts:1.Preparation of metal nanoparticles@polydopamine and application in peptides analysisIn this chapter,two kinds of core-shell composite nanoparticles of AuNPs@PDA and AgNPs@PDA were designed and synthesized by the mild redox polymerization of dopamine on the surface of metal nanoparticles under ambient conditions.This composite served as a novel matrix for the detection of small peptides by matrixassisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).This composite material showed free matrix background interference and increased signal intensity compared to the bare metal nanoparticles without PDA coating.Subsequently,AuNPs@PDA-Ti4+and AgNPs@PDA-Ti4+were prepared through the chelation reaction of hydroxyl groups on the PDA layer with Ti4+.The specific binding between AuNPs@PDA-Ti4+and phosphate was verified by using 4nitrophenylphosphoric acid di(tri)phosphate(pNPP)as the target molecule.Finally,AuNPs@PDA-Ti4+and AgNPs@PDA-Ti4+chelators were successfully employed to the selective enrichment of phosphopeptides for MALDI-TOF MS analysis.2.Study on the properties and mechanism of MoS2 as a MALDI matrixMolybdenum disulfide(MoS2)nanosheets were prepared via lithium ion intercalation,and were utilized for the first time as a matrix for the detection of small molecules by MALDI-TOF MS.Compared to commercial organic matrices and graphene matrix,the use of MoS2 matrix has the advantage of uniform matrix layers,low matrix background interference,and increased signal intensity in the analysis of amino acids,peptides,and fatty acids.A systematic comparison of positive and negative ion modes reveled that the MoS2 matrix only produced the corresponding deprotonated ion peaks in negative ion mode,which was rather different from the complex multiple alkali metal peaks present in the positive ion mode.Meanwhile,the mass spectrum obtained in the negative ion mode has lower background and better signal reproducibility.The ionization mechanism of MoS2 as matrix in negative-ion mode was further discussed.The deproton peak intensity of the fatty acid was reduced by adding a hole scavenger KSCN to the MoS2 matrix.It is indicated that the ionization of fatty acids is caused by the Auger recombination effect and electron injection from MoS2.The good reproducibility allowed the semi-quantitative analysis of sulfonamides.Furthermore,the quantitative MS analysis of sulfadimethoxine in human serum samples by the internal standard method also shows good performance,which satisfies the requirement for evaluation of the drug level in patients' serum and finally can realize convenient drug therapy monitoring.This work expands a new application branch for MoS2 and provides an alternative strategy for small molecule drugs analysis.3.MoS2/Ag nanohybrid:A novel matrix with synergistic effect for small molecule drugs analysis by negative-ion MALDI MSThis work reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles(MoS2/Ag)hybrid and its use as an effective matrix in negative ion MALDI-TOF MS.The nanohybrid exerts a strong synergistic effect,leading to high performance detection of small molecule analytes including amino acids,peptides,fatty acids and drugs.The enhancement of laser desorption/ionization(LDI)efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption,better dispersibility,increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS2.Moreover,both Ag nanoparticles and the edge of the MoS2 layers function as deprotonation sites for proton capture,facilitating the charging process in negative ion mode and promoting formation of negative ions.As a result,the MoS2/Ag nanohybrid proves to be a highly attractive matrix in MALDITOF MS,with desired features such as high desorption/ionization efficiency,low fragmentation interference,high salt tolerance,and no sweet-spots for mass signal.These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum.This work demonstrates for the first time the fabrication and application of a novel MoS2/Ag hybrid,and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.4.Disposable MoS2-arrayed MALDI MS chip for high-throughput and rapid quantification of sulfonamides in multiple real samplesIn this work,we demonstrate,for the first time,the development of a disposable MoS2-arrayed MALDI MS chip combined with an immunoaffinity enrichment method for high-throughput,rapid,and simultaneous quantitation of multiple sulfonamides(SAs).The disposable MALDI MS chip was designed and fabricated by MoS2 array formation on a commercial indium tin oxide(ITO)glass slide,A series of SAs were analyzed,and clear deprotonated signals were obtained in negative-ion mode.Compared with MoS2-arrayed commercial steel plate,the prepared MALDI MS chip exhibited comparable LDI efficiency,providing a good alternative and disposable substrate for MALDI MS analysis.Furthermore,internal standard(IS)was previously deposited onto the MoS2 array to simplify the experimental process for MALDI MS quantitation.96 sample spots could be analyzed within 10 min in one single chip to perform quantitative analysis,recovery studies,and real foodstuff detection.Upon targeted extraction and enrichment by antibody conjugated magnetic beads,five SAs were quantitatively determined by the IS-first method with the linear range of 0.5-10 ng/mL(R2>0.990).Good recoveries and repeatability were obtained for spiked pork,egg,and milk samples.SAs in several real foodstuffs were successfully identified and quantified.The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.5.Immunoaffinity microfluidic chip-mass spectrometry for on-line extraction and quantitative analysis of quinolones in milk samplesThe extensive use of quinolone(QNs)in veterinary has promoted the accumulation of their residues in foods derived from animals,which may lead to health concerns or potential antibiotic resistance.To help protect consumers from exposure to these potentially harmful compounds and control these chemicals in milk,many countries have established strict regulations,including specific maximum residue limits(MRLs)for the QNs.Currently there is a need for a rapid screening method for the detection of veterinary drugs in milk.In this work,an integrated microfluidic platform with multifunction coupled to mass spectrometry was developed for qualitative and quantitative analysis of seven different regulated QNs in milk samples.Sample extraction,immunoaffinity enrichment,magnetic separation,and online elution were performed simultaneously on the specifically designed device.Based on the specificity of antibodies,direct ESI MS at full scan mode without LC separation and further tandem mass spectrometry(MS/MS)analysis were developed for the identification of target QNs.One single isotope IS method was presented for quantitative analysis of seven QNs.Upon targeted online extraction and enrichment by antibody conjugated magnetic beads,seven QNs were quantitatively determined by the IS method with the linear range of 0.2/0.5-10 ng/mL(R2>0.991).The limits of detection(LODs)for the seven QNs were in the range of 0.047-0.490 ng/mL.The proposed immunoaffinity microfluidic chip-mass spectrometry is suitable for high-throughput screening of of multiple QNs in milk.