The Method Of Monitor And Assess Kidney Transplant Function:(Surface-Enhanced Raman Spectroscopy)
Mar 26, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
PART Ⅰ:Correlation of surface‑enhanced Raman spectroscopic fingerprints of kidney transplant recipient urine with kidney function parameters
Zhongli Huang, Shijian Feng & et al.
Routine monitoring of kidney transplant function is required for standard care in post-transplantation management, including frequent measurements of serum creatinine with or without kidney biopsy. However, the invasiveness of these methods with the potential for clinically significant complications makes them less than ideal. The objective of this study was to develop a non-invasive tool to monitor kidney transplant function by using Surface-Enhanced Raman Spectroscopy (SERS). Urine and blood samples were collected from kidney transplant recipients after surgery. Silver nanoparticle-based SERS(Surface-Enhanced Raman Spectroscopy) spectra of the urine were measured and evaluated using partial least squares (PLS) analysis. The SERS(Surface-Enhanced Raman Spectroscopy) spectra were compared with conventional chemical markers of kidney transplant function to assess its predictive ability. A total of 110 kidney transplant recipients were included in this study.PLS results showed significant correlation with urine protein(R2=0.4660, p<0.01),creatinine(R2=0.8106,p<0.01), and urea(R2=0.7808,p<0.01).Furthermore, the prediction of the blood markers of kidney transplant function using the urine SERS(Surface-Enhanced Raman Spectroscopy) spectra was indicated by R2=0.7628(p<0.01)for serum creatinine and R2=0.6539 for (p<0.01)for blood urea nitrogen. This preliminary study suggested that the urine SERS(Surface-Enhanced Raman Spectroscopy) spectral analysis could be used as a convenient method for the rapid assessment of kidney transplant function.
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Kidney transplantation represents a gold standard treatment for patients with end-stage renal disease(ESRD)!. Donor and recipient factors, surgical as well as immunologic factors may all contribute or cause the impaired graft function either immediately following the transplantation operation, termed delayed graft function, or at äny other time points after transplantation, Allograft dysfunction is typically revealed by serial laboratory tests showing rising serum creatinine(SCr), along with other potential findings including decreased urine volume, and an increase in blood pressure, pyuria and proteinuria°, Acute rejection (AR), acute tubular necrosis(ATN)and cyclosporine/tacrolimus toxicity are some of the more common etiologies of kidney transplant dysfunction7-9. If these issues are not properly identified and addressed, they may lead to permanent deterioration of kidney transplant function. Thus, patients with kidney transplants need routine monitoring of their kidney transplant function as a part of post-transplantation management10-1.
Conventional methods for evaluating the functional status of transplanted kidneys include clinical assessment of the patients based on volume status and urine output, serial SCr measurements with estimated glomerular filtration rates (eGFR), and even isotope-based examinations in rare circumstances2,13. If AR is suspected, renal transplant ultrasounds are often performed followed by transplant biopsy to confirm a pathologic diagnosis for the decision of the treatment plan. The procedure of the biopsy can be uncomfortable for patients and can lead to clinically significant complications, such as bleeding or allograft loss in the most extreme cases. Therefore, a non-invasive clinical tool that accurately determines the kidney transplant function is definitely desirable from both a patient and clinician perspective.
what is cistanche used for: improve kidney function
Raman spectroscopy (RS) is a specialized and non-invasive tool that emits an incident laser light through a fluid sample, resulting in scattered photons that produce a shift in a wavelength-dependent on the chemical composition of the fluid'4-17. The intensity of Raman scattering is proportional to the magnitude of the change in molecular polarization. Previously, the application of RS(Raman spectroscopy) was limited because of its low sensitivity, high costs, and lack of accessible, on-site analysis8. The recent technological advance of RS has brought forth a readily available, portable, and relatively cheap RS(Raman spectroscopy) device9. Surface-Enhanced Raman Spectroscopy(SERS)is a subtype of RS. designed specifically to increase its sensitivity of low concentration analytes, in which the treatment of a sample with an enhancement material significantly amplifies the electromagnetic fields of adsorbate molecules that are generated by the excitation of localized surface plasmons (LSP)20. Several SERS(Surface-Enhanced Raman Spectroscopy) enhancement materials have been described(e.g. colloidal metals and roughened metals, including gold, silver, and copper). These metal enhancers can increase the intensity of the Raman signal up to 10to lO°fold2. Our previous study demonstrated that our homemade colloidal silver nanoparticles(Ag NP)are a reliable and reproducible enhancement material for SERS(Surface-Enhanced Raman Spectroscopy). The objective of the current study was to evaluate the feasibility of using Ag NP-based SERS(Surface-Enhanced Raman Spectroscopy) of patients' urine samples to determine the kidney transplant function as compared to its conventional biochemical markers.
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METHOD AND ANIMAL
Specimen and data collection. Both urine and blood samples were collected from kidney transplant patients in the Department of Urology of West China Hospital in Sichuan University. These samples were collected concurrently within 3 days of the kidney transplant surgery. As following a standard operating procedure, the blood samples were sent to the clinical biochemistry laboratory in the West China Hospital for SCr and blood urea nitrogen (BUN) determination. The urine samples were collected by the patients themselves by using a 50 mL sterile tube. The urine samples were then delivered to the hospital laboratory within 8 h after collection. The urine tubes were centrifuged at 1500×g for 10 min to remove the cells and debris, and only the resultant supernatants were stored in aliquots at -80 °C refrigerator prior to use. The levels of protein and creatinine in the urine samples were determined in the Clinical Chemistry Laboratory at Vancouver General Hospital (Vancouver, British Columbia, Canada).
Silver (Ag) nanoparticles and urine sample preparation. Ag colloids were prepared using hydroxylamine hydrochloride and Ag nitrate as described previously23,24. Briefly, the glassware used in the following procedures was cleaned with chromic acid lotion, and then rinsed thoroughly with milli-O water before use., 4.5 mL of 0.1 M NaOH solution was rapidly added to 5 mL of6×10-2M hydroxylamine hydrochloride solution, and the mixture was stirred until a homogeneous suspension was formed. The resultant solution was mixed with 90 mL of 1.11×10-3M AgNO, aqueous solution at room temperature until the presence of a milky-grey color. This Ag colloid suspension was then stored in the dark before use. In order to ensure the consistent shape and size of the Ag NP, all the experimental steps and processes were standardized. Once ready for use, the Ag colloids were pelleted by centrifugation at 10,000×g for 10 min. A transmission electron microscopy (TEM) image of Ag nanoparticles was taken with the sizes of the synthesized Ag NP following a normal distribution with a mean diameter of 35±5 nm (Suppl. Figure 1). The pelleted Ag NP were then mixed with urine samples at a 1:1 ratio in a rectangle aluminum plate（10 μL total volume）. The same batch of Ag NP was used for one round of SERS(Surface-Enhanced Raman Spectroscopy) measurement of all of the urine samples at the same time to avoid the probable inconsistency of the Ag NP. The repeated measurements(technical replicates)of the same samples were performed using the same or a different batch of the Ag NP. The Ag colloid/urine mixture was then air-dried at room temperature for 60 min prior to SERS(Surface-Enhanced Raman Spectroscopy) measurement to facilitate molecular aggregation. If the prepared samples displayed the so-called "coffee ring effect" or overt crystal formation(few cases), they were re-made until the absence of this problem. Of note, aggregation agents are believed to increase the signal intensity of SERS(Surface-Enhanced Raman Spectroscopy), however, we elected to forego the use of such agents in hopes of streamlining the sample preparation process.
cistanche extract benefit: prevent kidney diseases
SERS(Surface-Enhanced Raman Spectroscopy) measurement. SERS spectra of the Ag colloid/urine mixture were measured with a commercial Raman spectrometer(AURA, Verisante Technology, Vancouver, BC, Canada) equipped with a 785 nm diode laser for Raman excitation, similar to the prototype reported previously by Zhao et al.25,26.The laser beam was delivered to the sample through a single fiber with a core diameter of 200 μm. Raw signals were collected by a probe consisting of a central excitation fiber surrounded by one hundred and ten 100-um core-diameter collection fibers and then delivered to the spectrometer. Each SERS(Surface-Enhanced Raman Spectroscopy) spectrum was acquired with 150 mW excitation laser power and an integration time of one second with a spectral resolution of 8 cm-lin in the wavenumber range of 500-1800 cm-1. For each dried droplet or sample, three spectra were measured with spotting the whole sample area on the aluminum slide, and the mean was used in subsequent data analysis. A monitor was also used to minimize the heterogeneity of each test of the same sample by visualizing the spectrum simultaneously during the measurement. If the visualized spectrum was largely different from each other, another 3-6 spectra were then obtained until the consistency was achieved.
Data analysis. The resultant SERS(Surface-Enhanced Raman Spectroscopy) spectrum of the urine samples in the wavelength range of 500-1800-1 cm was extracted by subtracting the fluorescence background using the fifth-order polynomial fitting algorithm27. Partial least squares(PLS) regression with leave-one-out cross-validation (LOO-CV) of the whole spectra was used to quantify the biochemical parameters of kidney transplant function including urine protein, urine creatinine(UCR), urine urea, SCr, and BUN. The concentrations of these parameters obtained from blood/urine biochemistry analysis were used as a gold standard to compare the SERS(Surface-Enhanced Raman Spectroscopy) urine analysis. TIBCO STATISTICA software (Version 10, TIBCO SOFTWARE Inc., Palo Alto, CA, USA) was used for PLS regression analysis.
Ethical approval. This study(Approval Number: 2019(748))was approved by the ethics committee at West China Hospital of Sichuan University, and all participants signed the informed consent prior to sample collection. Sichuan University and West China Hospital's ethics committee confirmed that all the research activities were performed in accordance with relevant guidelines and regulations.
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RESULT
A total of 110 kidney transplant recipients(80 male and 30 female; mean age of cohort 41.5 years) participated in this study. The urine biochemistry results revealed that the range of urine protein was 0.05-6.83 g/L, UCR 1.03-22.49 mmol/L, and urine urea 29.6-535.8 mmol/L, the biochemical results of blood tests indicated that the range of SCr was 39-1461 umol/L and BUN 3.2-25.1 mmol/L. The demographic characteristics of patients and their urine and blood test results were summarized in Table 1.
Table 2. Summarize previous reported SERS(Surface-Enhanced Raman Spectroscopy) peak positions and vibration mode assignments1.
The SERS(Surface-Enhanced Raman Spectroscopy) spectra of Ag NP/ urine mixtures were measured under the same conditions(1 day after mixture preparation). The SERS(Surface-Enhanced Raman Spectroscopy) spectra of samples revealed many dominant vibration bands, implying a tight interaction between the Ag colloid and urine molecules resulting in the signal enhancement. As shown in Fig.1, the main SERS(Surface-Enhanced Raman Spectroscopy) peaks (vibrational assignments) of urines were assigned to 1285,1004, 702, and 1383 cm-1, which biochemically corresponds to cytosine, urea, cholesterol, and CH3 band, respectively8. Other biochemical assignments of the SERS(Surface-Enhanced Raman Spectroscopy) peaks shown in Fig.1 are summarized in Table 2. To explore if the SERS(Surface-Enhanced Raman Spectroscopy) could be used as a non-invasive tool to assess the status of kidney transplants, PLS generated models were utilized to predict the concentrations of kidney transplant function markers in the urine and blood. Figure 2 showed the results of PLS analysis of the urine protein, UCR, and urine urea as compared to the SERS(Surface-Enhanced Raman Spectroscopy) spectra, respectively. As a reference for the prediction ability of PLS analysis, R'>0.67 indicated the high predictive accuracy, Range of0.33-0.67 the moderate predictive accuracy, R' range of 0.19-0.33 the low predictive accuracy, and R'<0.19 unacceptable variables2. The urine results revealed an R²=0.4660（moderate） for urine protein（p<0.001）， R²=0.8106（high）for UCR（p<0.001）， and R²=0.7808（high） for urine urea（p<0.001）. Figure 3 showed the results of PLS analysis of the blood SCr and BUN as compared to the urinary SERS(Surface-Enhanced Raman Spectroscopy) spectra, respectively. The blood results revealed an R²=0.7628（high） for SCr （p<0.001）and R²=0.6539 （moderate to high）for BUN（p<0.001）. Because the low concentrations of kidney transplant function biomarkers—normal transplant function are not a concern in clinical care, we calculated the standard deviation (SD)of their high concentrations according to the borderline level. For the urine protein<0.3 g/L, the calculated SD was 0.33 g/L, and>0.3 g/L, the SD 0.44 g/L.For the UCr<7 mmol/L, the calculated SD was 1.69 mmol/L, and>7 mmol/L, the SD 1.64 mmol/L.For the urine urea<266 mmol/L, the SD was 47.88 mmol/L, and>266 mmol/L, the SD29.57 mmol/L, The serum biomarkers are mainly used for the kidney transplant function in the clinical practice. For the SCr<110 mmol/L, the SD was 87.98 mmol/L,and>110 mmol/L, the SD 108.21 mmol/L.For the BUN<7.7 mmol/L, the SD was 2.15, and>7.7 mmol/L, the SD 2.32 mmol/L.
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| Figure 2. The PLS analysis generated models were used to predict concentrations of molecules in the urine. PLS analysis for urine protein (a), UCR (b), and urine urea (c). Te results demonstrated that R2=0.4660 for urine protein (a) (p<0.001), R2=0.8106 for UCR (b) (p<0.001), R2=0.7808 for urine urea (c) (p<0.001). | Figure 3. The PLS analysis generated models were used to predict the concentrations of blood biomarkers for kidney transplant function. The results showed that R2=0.7628 for SCr (a) (p<0.001) and R2=0.6539 for BUN (b) (p<0.001), respectively. |
