The Construction Of The Setups Of Optical Tweezers Assisted Suspension Bead Based Biodetection And Their Applications

Bioaffinity assay is a very important part of life science research and plays a vital role in clinical diagnosis,drug development,disease-related research and other fields.Being easy to operate,highly sensitive and able for microvolume detection are the new requirements that modern life science research put forward on bioaffinity assay technology.In recent years,suspension bead-based assay technology is widely used for its high-throughput and microvolume detection characteristics,but it detects the fluorescence signal emitted by microspheres for a short time in the flowing fluid,not the fluorescent image of the bead,thus greatly limits its sensitivity.In addition,the existing trapping technologies used to assist the fluorescence imaging of the beads cannot accurately capture the microspheres.Fortunately,optical tweezers is a precise and non-invasive means of particle trapping,and can flexibly and accurately control the capture position of beads in the sample chamber,by virtue of these advantages optical tweezers technology has a wide range of applications in a number of disciplines.Based on the above background,this dissertation proposes the idea of applying optical tweezers to the detection of signal of suspension bead-based bioaffinity assay.It is committed to developing a new type of beads based detection technology to realize efficient,accurate and rapid detection of biomolecules in microvolume sample.The main work carried out by this dissertation is summarized as following:(1)The setups of a single-trapping optical tweezers and a multiple-trapping optical tweezers based on an inverted fluorescence microscope platform were constructed,and the optical path construction and trapping characteristics of the two setups were studied and discussed in detail.The following details were investigated:the selection of the specifications of the optical setup components,the modulation of the beam characteristics in the optical path of the two setups of optical tweezers,the relative position between the trapping plane and the imaging focal plane of the objective lens,the comparison of the trapping ability between the two optical tweezers capturing of beads with different size.The results show that the single-trapping optical tweezers can stably capture the particles with particle size between 1 ?m and 10 ?m,and the multiple-trap optical tweezers can capture particles with particle size between 1 pm and 5?m.Therefore,the microspheres with particle size of 3 ?m and 5 ?m were selected as the assay beads with the stability of trapping and the ease of imaging signal analysis.Finally,a microfluidic chip was used to construct the measuring of the trapping stiffness of the optical tweezers,and the data show the linearity between the optical trapping stiffness and the laser power.(2)A suspension bead-based assay for the two kinds of retrovirus DNA of avian influenza virus H7N9 using the single optical tweezers with a blue laser was successfully developed.The CdSe/ZnS quantum dots of 605 nm emission wavelength were selected as fluorescent labeling material.The synchronous excitation of 605 nm quantum dots fluorescence using the same blue laser was realized while the beads were trapped,and 3 ?m and 5 ?m microspheres for size encoding were used as the carrier of the suspension bead-based assay to attain simultaneous detection of DNA sequences of the two-component virus genes.The microspheres were accurately captured and imaged in the sample chamber.The results show that the limit of detection of the two genes reaches 1.0 pM and 1.2 pM,respectively,indicating high sensitivity of the two targets,and the linear range for both targets is three orders of magnitude showing a wide linear range for detection.The two kinds of beads were tested for non-target nucleic acid sequence and the data show good selectivity,specificity and good anti-interference ability.Thus,this method can be used for trace virus gene nucleic acid sequence quantitative detection and classification,and is highly potential in the early detection and prevention of infectious diseases.(3)A novel 3x3 array multiple-trap near-infrared optical tweezers was constructed,and a suspension bead-based assay for the detection of dual targets of prostate-specific antigen(PSA)(fPSA and tPSA)in human serum was developed.The multiple-trap optical tweezers was constructed using a diffractive optical element(DOE)and the results show that the optical configuration is simple,and the energy utilization rate of the laser is high,moreover,the capture range in the view-field is much larger than that of the single optical trap,so the multiple-trap quickly captures the beads to form the array and improves the detection speed and throughput.Quantum dots with 605 nm emission was used as the fluorescent labeling material,and a fiber-coupled 405 nm laser was applied for wide-field excitation of the fluorescence to obtain a stable and clear fluorescence image of the 3x3 bead array.Using the size encoding of suspended microspheres,the determination of PSA dual targets was realized.In the 5%serum environment,the detection limits are 3.8 pg/mL for fPSA and 2.5 pg/mL for tPSA,respectively.The linear ranges of fPSA and tPSA are 3 orders of magnitude and nearly 4 orders of magnitude,respectively.It is shown that there is no significant change of signal intensity for each microsphere between single beads system and mixed beads system,indicating that the mixed beads with different size can be used for the detection of the dual targets of PSA in complex fluid samples.The coexistig interfering proteins exhibit no obvious signal response,indicating that this method has excellent anti-interference ability.This method was applied to the detection of real serum samples and good results were obtained.The above results demonstrate that the setup and the detection approaches developed in this work are highly potential in diagnostic and prognostic study of human diseases such as cancers.In this dissertation,the stable and accurate fluorescence imaging of suspended beads is realized by using optical tweezers.How the suspended beads can be fixed in liquid phase system for bead based assay has been challenging.This work shows a novel approach to solve this problem to simplify the detection steps and improve the detection effectiveness.The detection method of multi-component was realized by the size coding of microspheres,and the accurate and sensitive detection of viral nucleic acid and cancer marker and other biomolecules was realized.The results show great potential of this method in biomedical applications.