| Pregnancy of unknown location and pregnancy of uncertain viability has attracted the attention of doctors,because some miscarriages and ectopic pregnancy are hidden in the stage.Therefore,it is of great significance to predict the adverse outcome of early pregnancy,improve the detection rate of adverse pregnancy,help to be treated as early as possible,and reduce the complications and mortality caused by ectopic pregnancy.In general,the speed of serum β-h CG doubling,the level of progesterone and ultrasonography are the commonly methods to judge the early adverse pregnancy.However,the existing prediction methods have some limitations.On the one hand,the sensitivity and specificity of prediction are not ideal,and the waiting time is relatively long,which requires repeated blood sampling and follow-up for ultrasound examination,all of which increase the cost and anxiety,while the waiting process may have serious complications.β-h CG can be detected not only in maternal serum but also in urine and cervical secretions.Sensitive and specific detection of β-h CG in maternal cervical secretions and serum is of vital significance for early pregnancy evaluation.However,the accurate detection of the trace amount of β-h CG in cervical secretions is a great challenge because of its low level.Among various bioassay methods,time-resolved fluoroimmunoassay based on lanthanide(Ln3+)chelates has been adopted as one of the most sensitive bioassay methods for β-h CG detection.The time-resolved photoluminescence(TRPL)technique can distinguish the long-lived PL of Ln3+(from μs to ms)from the short-lived background noise(in the ns range)which arise from biological autofluorescence and scattered light.By taking advantage of the intense and long-lived PL of the nanoprobes,we can completely eliminate the background autofluorescence.In this thesis,a unique strategy is proposed for β-h CG detection in a heterogeneous sandwich-type bioassay by using Li Lu F4:Ce/Tb nanoparticles as TRPL nanoprobes.It provides remarkably improved detection sensitivity in bioassay than conventional steady-state fluorescence,so that the detection of β-h CG can be more sensitive than commercial β-h CG assay kit.The ratio of β-h CG in cervical secretion and serum can be used as an indicator to predict the adverse pregnancy outcome of pregnancy of unknown location and uncertain viability,which is expected to shorten the time window of adverse pregnancy judgment and reduce the cost of blood test and anxiety of waiting for ultrasound results.It also can provide evidence for clinical intervention in advance and avoid serious complications caused by delayed diagnosis.The main methods and results of this paper are as follows:1.Synthesis of oleate-capped Li Lu F4:Ce/Tb Nanoparticles(1)Oleate-capped Li Lu F4:Ce/Tb nanoparticles were synthesized via a thermal decomposition method.(2)Powder X-ray diffraction(XRD)pattern of the nanoparticles was collected by an X-ray diffractometer(Mini Flex2,Rigaku)with Cu Kα1 radiation(λ = 0.154187 nm).XRD pattern of the nanoparticles displays intense diffraction peaks that could be well indexed into tetragonal Li Lu F4(JCPDS No.027-1251),indicating pure phase and high crystallinity of the resulting nanoparticles.(3)Both the low-and high-resolution transmission electron microscopy(TEM)images were acquired by using a TECNAI G2 F20 TEM equipped with the energy dispersive X-ray spectrum(EDS).TEM image showed that the nanoparticles are rhomboid with a mean size of(34.5 ± 1.3)×(29.5 ± 1.3)nm.HRTEM image exhibitted clear lattice fringes with an observed d spacing of 0.46 nm The as-synthesized nanoparticles were hydrophobic owing to oleate ligands capping on their surface,which agreeed well with the lattice spacing of the(101)plane of tetragonal Li Lu F4.(4)According to TEM observation results,200 nanoparticles were selected to calculate the average size.(5)Selected area electron diffraction(SAED)pattern of Li Lu F4:5%Ce/5%Tb nanoparticles,showing high crystallinity of the nanoparticles.(6)Compositional analysis through EDS revealed the host elements of Lu and F and the dopants Ce and Tb in the nanoparticles.(7)By monitoring the Tb3+ emission at 544 nm,a broad excitation band arising from the4f→5d transition of Ce3+ at 296 nm was observed,suggesting an efficient energy transfer from Ce3+ to Tb3+.PL decay curve of the nanoparticles shows a single-exponential decay with a fitted PL lifetime of 4.69 ms.(8)The absolute photoluminescence quantum yield(PLQY)of the nanoparticles was determined to be 67.6 ± 0.8%.2.Synthesis of biotinylated Li Lu F4:Ce/Tb Nanoparticles and luminescent properties test(1)We rendered the hydrophobic nanoparticles hydrophilic through an acid washing treatment,which yielded ligand-free nanoparticles with nearly unchanged particle size and morphology.As a result,these ligand-free nanoparticles can be well dispersed in water and are allowed for conjugating with various biomolecules such as biotin through electrostatic absorption or the strong chelation of Lu3+ exposed on the surface of the nanoparticles.(2)The FTIR spectra showed that the asymmetric and symmetric stretching vibrations of methylene(–CH2–)in the long alkyl chain peaking at 2923 and 2857 cm-1 and the vibrations of carboxyl(–COO–)peaking at 1551 and 1452 cm-1 in oleate-capped nanoparticles disappeared in ligand-free nanoparticles,while in biotinylated nanoparticles,a strong amide band centered at 1646 cm-1 is clearly observed.(3)The TGA curves of ligand-free and biotinylated nanoparticles displayed a weight loss of 1.0% and 1.5%,respectively,in marked contrast to that of 4.7% in oleate-capped nanoparticles with an obvious decomposition temperature at 340 °C.(4)Hydrodynamic diameter distributions and ζ potentials of ligand-free and biotinylated Li Lu F4:5%Ce/5%Tb nanoparticles dispersed in aqueous solution(p H 6.9).The hydrodynamic diameter of the nanoparticles increased from 24.4 nm in ligand-free nanoparticles to 68.2 nm in biotinylated nanoparticles due to the conjugation of biotin to the NP surface that may cause slight aggregation of the nanoparticles.The ζ potential changed from +33.9 m V in ligand-free nanoparticles to +13.7 m V in biotinylated nanoparticles,as a result of reduced positively-charged Lu3+ ions exposed on the surface of the nanoparticles.(5)Notably,the photoluminescence properties of the nanoparticles were significantly improved after removing the surface oleate ligands,because more excitation light can be absorbed by Ce3+ ions which can effectively sensitize Tb3+ instead of oleate ligands which cannot.The integrated photoluminescence intensities of ligand-free and biotinylated nanoparticles were enhanced by factors of 3.4 and 1.7,respectively,in comparison with their oleate-capped counterparts,with photoluminescence lifetimes of Tb3+ increasing from 4.69 ms in oleate-capped nanoparticles to 6.71 ms in ligand-free nanoparticles and 7.49 ms in biotinylated nanoparticles.3.Heterogeneous assay of β-h CG based on biotinylated Li Lu F4:Ce/Tb nanoparticles 1)The calibration curve shows that the photoluminescence intensity of the nanoprobes increases gradually with increasing the β-h CG concentration and exhibits a linear dependence with the β-h CG concentration in the range of 0–8 ng·ml-1.The limit of detection(LOD),defined as the concentration corresponding to three times the standard deviation above the signal measured in the control,was determined to be 6.1 pg·ml-1,which is about three order of magnitude improvement relative to that of commercial β-h CG assay kit based on chemiluminescence.2)By contrast,in nonbinding control where β-h CG antigen was replaced by other possible interfering biomolecules and electrolytes in women serum and cervical secretions including carbohydrates,proteins,and metal ions under otherwise identical conditions,the TRPL signal was nearly undetectable due to the lack of specific binding,indicative of a high specificity of the assay.3)To validate the applicability of Li Lu F4:Ce/Tb nanoprobes for real sample detection,we measured the β-h CG levels of three women serum samples and three cervical secretion samples upon addition of different concentrations of β-h CG standard solution and then evaluated the CVs and the recoveries of the assays.It was found that the CVs were below 9% and 10% and the recoveries were in the range of 91–108% and 93%–103% for the assays in serum and cervical secretion samples,respectively.These CV and recovery values are within the acceptance criteria(CVs ≤ 15%;recoveries in the range of 90%–110%)set for bioassay method validation,affirming high reliability and sensitivity of Li Lu F4:Ce/Tb TRPL nanoprobes for serum and cervical β-h CG detection.4.Determination of the ratio of cervical secretion to serum β-h CG by time-resolved immunofluorescence based on Li Lu F4:Ce/Tb nanoprobe and its evaluation of adverse pregnancy outcome(1)42 pregnant women in early pregnancy,diagnosed as pregnancy with unknown location and uncertain viability,were included in the study,clinical information was collected,test samples were collected,follow-up was conducted according to the requirements until the pregnancy outcome was determined,and they were grouped according to different outcomes.28 cases were viable intrauterine pregnancy,6 cases were miscarriage of intrauterine pregnancy and 8 cases were ectopic pregnancy.(2)The application of the novel TRPL nanoprobes was for accurate detection of β-h CG in cervical secretion samples and maternal serum.There were significant differences in the ratio of β-h CG levels in cervical secretions and serum(β-h CG cervical/serum),the 48 hour growth rate of human serum β-h CG and the value of progesterone in different pregnancy outcomes.The ratio of β-h CG cervical/serum was then correlated with the pregnancy outcomes(viable intrauterine pregnancy,miscarriage of intrauterine pregnancy,or ectopic pregnancy).In the viable group of intrauterine pregnancy,the β-h CG levels in cervical secretion were about 3–5 orders of magnitude lower than those in serum,while in the groups of miscarriage of intrauterine pregnancy and ectopic pregnancy,the β-h CG levels in cervical secretion increased significantly and were 1–2 orders of magnitude lower or even higher than those in serum.These observations suggest that ratio of β-h CG cervical/serum can be a valid indicator of early pregnancy viability in unknown location.(3)From the ROC curve,there were differences in the prediction of different pregnancy outcomes among different indicators,but the ratio of β-h CG cervical/serum was larger than that under the curve of other three indicators,indicating that the prediction value of this indicator was higher.(4)Distribution of ratio of β-h CG cervical/serum of 42 pregnant women with days of gestation from 30 to 48 and pregnancy outcomes of viable intrauterine pregnancy,miscarriage of intrauterine pregnancy,and ectopic pregnancy.The ratio of β-h CG cervical/serum was higher than 0.003 results in an ectopic pregnancy or miscarriage while that was lower than 0.003 leads to a viable intrauterine pregnancy.(5)According to the review of adverse pregnancy outcome,the earliest occurrence of abnormal ratio was 33 days of menopause,which suggested that this value might shorten the window period by 1-2 weeks as a predictive index.In summary,the main conclusions of this study can be summarized as follows:1.The Li Lu F4:Ce/Tb nanoparticles exhibit excellent luminescent properties with a high photoluminescence quantum yield and a long photoluminescence lifetime.2.By conjugating with biotin,the nanoparticles can be functionalized as efficient TRPL nanoprobes for sensitive and specific detection of β-h CG antigen in a heterogeneous sandwich-type bioassay.In our study,the linear range is of 0-8 ng·ml-1,and the detection is limit down to 6.1 pg·ml-1.3.By comparing the results with their pregnant outcomes,it was unveiled that the ratio of β-h CG cervical/serum can be a good indicator of early pregnancy viability in unknown location4.This method does not need repeated blood sampling examination.It can be expected to shorten the time window of bad pregnancy judgment,provide basis for clinical intervention in advance,and avoid serious complications caused by delayed diagnosisAbove all,these findings bring new opportunities into perinatal medicine.By employing the Li Lu F4:Ce/Tb TRPL nanoprobes,β-h CG detection of different body fluid can be applied in clinical application.The result of our study also lays a foundation for future development of luminescent nanoprobes for versatile biomedical applications. |
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