BÁO CÁO KHOA HỌC: "Sử dụng cảm biến sinh học là vi khuẩn phát sáng đã biến đổi gen để khảo sát nhanh hàm lượng asen trong nước ngầm"

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Ô nhiễm Asen (thạch tín) trong nước uống bắt nguồn từ nước ngầm được phát hiện tại nhiều khu vực trên thế giới, nhất là tại các nước có mật độ dân cư cao như Ấn độ, Băng la đet, Trung quốc và Việt nam. Để nhằm mục tiêu giảm thiểu nhiễm độc Asen cho cộng đồng dân cư thì một trong những bước quan trọng nhất là xác định sự ô nhiễm tại từng giếng càng sớm càng tốt.

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Nội dung Text: BÁO CÁO KHOA HỌC: "Sử dụng cảm biến sinh học là vi khuẩn phát sáng đã biến đổi gen để khảo sát nhanh hàm lượng asen trong nước ngầm"

Sử dụng cảm biến sinh học là vi khuẩn phát sáng đã biến đổi gen để khảo sát nhanh hàm lượng asen trong nước ngầm Ô nhiễm Asen (thạch tín) trong nước uống bắt nguồn từ nước ngầm được phát hiện tại nhiều khu vực trên thế giới, nhất là tại các nước có mật độ dân cư cao như Ấn độ, Băng la đet, Trung quốc và Việt nam. Để nhằm mục tiêu giảm thiểu nhiễm độc Asen cho cộng đồng dân cư thì một trong những bước quan trọng nhất là xác định sự ô nhiễm tại từng giếng càng sớm càng tốt. Kỹ thuật mới sử dụng cảm biến sinh học là vi khuẩn để xác định nhanh hàm lượng asen trong nước ngầm có triển vọng hỗ trợ cho các phương pháp phân tích truyền thống do các phương pháp phân tích hiện trường hiện nay có độ chính xác không cao. Trong nghiên cứu này cảm biến vi khuẩn phát sáng Escherichia coli DH 5 (pJAMA8-arsR) đã được thí nghiệm để xác định asen theo qui trình tối ưu. Để tránh sự hấp thụ asen bởi các hydroxit sắt, các mẫu nước ngầm được axit hoá về pH 2 bằng HNO3 (nồng độ cuối cùng là 0,015M). Một lượng
tương đương giữa mẫu và vi khuẩn trong môi trường LB được trộn với nhau và được trung hoà lại bằng dung dịch pyrophophat (nồng độ cuối cùng là 5mM). Thử nghiệm với 194 mẫu nước ngầm tại Việt nam cho thấy giới hạn phát hiện của cảm biến sinh học này với các mẫu thực là 7 µg/l. Các phép đo có độ chính xác khá cao trong khoảng nồng độ 10-100µg/l ( với r2=0.9). Kết quả này vượt trội hơn so với các bộ kiểm tra hiện trường thông thường. Sai lệch âm và dương là 8.0% và 2.4% khi dựa trên tiêu chuẩn về hàm lượng asen trong nước ngầm của WHO ( 10µg/l) để xác định mẫu có hay không ô nhiễm asen. Độ chính xác cao của cảm biến sinh học thu được một phần nhờ các phép đo luôn được lặp lại ba lần. Tốc độ thí nghiệm nhanh và độ chính xác cao hứa hẹn sự ứng dụng rộng lớn của cảm biến sinh học vi khuẩn trong sàng lọc sự ô nhiễm asen trên diện rộng. 1. INTRODUCTION Arsenic is polluting the groundwater at many places around the world, like Bangladesh, West Bengal - India, Vietnam,
China, or Argentina, etc (Berg, 2001; Chakraborti, 2003 and Smedley 2002). Arsenic pollution is considered as the most serious natural worldwide calamity of the present moment. Around 150 million people in West Bengal and Bangladesh, and over 2 million in China are exposed to unacceptable health risks by consuming arsenic contaminated drinking water. A similar situation may be occurring in Vietnam, where arsenic is suspected to potentially contaminate the tube wells of around 13.5 percent of the Vietnamese population, some 10.5 million persons (Berg, 2001; UNICEF, 2002). Although a coarse picture on the distribution of arsenic exists in the groundwater in these affected areas, there are millions of individual tube wells yet remaining to be measured (Kiniburgh 2002, Chakraborti, 2003). Unfortunately, arsenic is very heterogeneously distributed spatially, and the arsenic contents in two nearby wells with 100 m in distance can be as different as from 10 to higher 300 µg/L (Berg, 2001, 2003; Smedley, 2002). It thus remains absolutely necessary for effective arsenic mitigation campaigns to screen every individual tube well (blanket
screening) and determine whether or not the quality of the potable water complies with current arsenic guideline values (for WHO 10 µg As/L, for Bangladesh currently 50 µg As/L). Considering the poor technical facilities in the most exposed countries, arsenic testing for a large number of wells poses an extreme challenge. So far, mostly the chemistry based commercial field test kits named Merck, Hach, Arsenator, ANN, or local imitations have been applied in Bangladesh, India, Vietnam and other countries (Kiniburgh, 2002). Unfortunately, chemical field kits have low precision, reproducibility and accuracy at arsenic concentrations between 10 µg/L and 100 µg/L. For example, among 290 wells tested both by field kits and flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS), take into account the samples with arsenic concentrations in the range of 50-100µg/L as high as 68% of the samples measured by the field kits scored false negative and 35% false positive (Rahman, 2002).
Quite a number of bacterial biosensors responsive to different target compounds have been designed in the past decade. Bacterial biosensors are genetically modified bacteria that produce a reporter protein (such as bacterial luciferase) in response to the presence of a target chemical. Luminescent bacterial biosensors for arsenic measurement have been developed recently as potential and promising alternative methodologies (Daunert, 2000; Tauriainen 2000; Petänen, 2002; Van der Meer, 2004). Luminescent bacterial biosensors for arsenite display a lower detection limit of around 4 µg/L As(III) in potable water with standard deviation of around ± 5%, which is more than sufficient to comply with regulatory guidelines (Stocker, 2003). Here a detailed protocol has been developed to measure arsenic concentrations in Vietnamese groundwater pumped from small-scale tube wells. The accuracy of the biosensor used to predict arsenic concentrations at the guideline level of 10 µg /L was determined by comparing with data
obtained simultaneously from AAS and AFS for the same samples. Our results provide the first larger scale screening of field samples with a biosensor-based test. 2. EXPERIMENTAL PROCEDURES 2.1. Groundwater sampling 194 groundwater samples from tube wells (family scale) were sampled in villages located at arsenic affected areas of the Red river and Mekong river delta, Vietnam. Groundwater was collected at the tube by hand or electrical pumping. Water was taken after 10 minutes pumping, when the oxygen concentration in the water reached a stable value, which was measured online by using a dissolved oxygen electrode (PX 3000, Mettler-Toledo). 50mL groundwater samples were filtered through 0.45µm filter paper and transferred to acid-washed plastic bottles. Samples were acidified to pH about 2 by addition 0.1mL HNO3 (7.5M, Merck) to final concentration of 0.015M.
Water bottles were transfer to the lab, stored at 4oC and analysed for arsenic in two weeks. 2.2. Arsenic measurement by AFS and AAS Arsenic in the groundwater samples was measured in parallel by using an AAS-6800 (Shimadzu, Japan) at CETASD’s laboratory, Hanoi University, Vietnam and an AFS Millenium Excalibur (PS Analytical Ltd, Kent, U.K.) at EAWAG, Switzerland. Calibration solutions were prepared by using a stock solution of 1000 mg As(III)/L (J.T Baker, Netherlands) and deionised water. Calibration curves were established with final concentrations of 0, 1, 2, 4, 8 and 10 µg As/L (about 0, 0.013, 0.027, 0.053, 0.107 and 0.13 µM respectively). The data obtained by the two methods were used to validate the Vietnamese AAS- method, which was subsequently used to validate the biosensor test. Standard reference materials as SPS-SW2 standard (Spectra pure Standard-Norway) and ICP Multi element standard VI (Merck) were used to check the accuracy of AAS and AFS methods.
2.3. Arsenic measurement by genetically modified E. coli DH5 (pJAMA-arsR) biosensor The arsenic biosensor was E. coli DH5 (pJAMA-arsR), which was used under the cultivation and storage conditions as described previously (Stocker, 2003). Briefly, arsenite determination by the bacterial biosensor is based o- n bioluminescence light produced by the cells in response to arsenite contact. The intensity of the bioluminescence is proportional to the arsenite exposition and can be recorded after predefined incubation periods in a luminometer. Biosensor cells carry a plasmid with the genes for bacterial luciferase (luxAB) under expression control of the ArsR transcriptional repressor protein. Cellular entrance of arsenite causes release of transcriptional repression and subsequent synthesis of luciferase by the cells. Arsenate is spontaneously reduced by the cells to arsenite and hence can also indirectly cause derepression and luciferase synthesis (Daunert, 2000; Rosen, 2002; Stocker 2003,). Biosensor assays were conducted in 4 ml sterilised glass
vials. The bacteria suspension was prepared just before the assay by mixing a 1.3 mL frozen aliquot of biosensor cells with 10 mL sterilised Luria-Broth (LB) medium. Equal amounts of aqueous sample and cell suspension (500µL) were pipetted per vial, vials covered with a screw cap and incubated on a rotary shaker at 200 rpm and 30°C. After 90 minutes, 50 µl of n-decanal solution (18 mM in a 1:1 v/v ethanol-water solution) was added to the vials as substrate for the luciferase reaction. Light emission was recorded after 3 minutes in a luminometer (Junior-Berthold, Germany) and is expressed as relative light units (RLU). Each sample was measured in triplicates, which were used to calculate the average light emission. The response to samples with unknown arsenic concentrations was compared to that of a standard series of arsenite concentrations, containing 0, 0.1, 0.2, 0.4, 0.8 and 1µM As (0, 7.5, 15, 30, 60 and 75 µg As/L) and prepared in arsenic- free groundwater. Arsenic concentrations in unknown samples were determined by linear interpolation of the standard curve. In case of acidified samples, 25 µL of a 200mM sodium pyrophosphate solution (Na4P2O7. 10 H2O,
Sigma) was added per 500µL groundwater sample in situ to the test vial. All experiments were carried with triple measurement and used for average calculation. 3. RESULTS AND DISCUSSION 3.1. The protocol for determination of As in groundwater by genetically modified E. coli DH5 (pJAMA-arsR) biosensor The most optimal combination found is acidification groundwater to pH 1.8-2.0 by addition of HNO3 to concentration of 0.015mM, the acidified groundwater sample then was mixed with LB solution contained bacterial biosensor by ratio 1:1, the suspension was subsequent added in situ pyrophosphate (5 mM final concentration) to readjust the pH to about neutral. This protocol was described at Figure 1 and subsequently followed by all field samples.
Figure 1: Flow chart for biosensor test 3.2. Rapid screening of field samples with the bacterial biosensor. Firstly the reference method for total arsenic determination
by AAS at CETASD in Vietnam was validated by comparison with the AFS method performed at the EAWAG in Switzerland on a set of 111 groundwater samples collected in Mekong river delta, Vietnam (Fig. 2). A linear correlation between the AAS and AFS data was calculated by regression analysis. Linearity with r2-values equal 0.99 were obtained, hence giving confidence that the AAS method at the CETASD would give a proper calibration for comparisons to the biosensor obtained values afterwards. Chemical compositions of groundwater at Vietnamese arsenic contaminated areas are quite variety as present at Table 1 (internal data). Arsenic, iron, bicarbonate, phosphate, ammonium, chlorite, etc concentrations are different as from 10 to 1000 times between sampling points. This hence is challenge for the application of biosensor as arsenic test device because living bacteria cells are used. Response of biosensor to dissolved arsenic in groundwater was checked using concentrations from 0 to 3 µM (0 - 225µg/L). The groundwater matrix is arsenic free
and 0.18mM of iron (20mg/L). As present in Figure 3a the curve was linear in the range of 0 to 1µM with r2-values equal 0.99, above this concentration bacteria response to arsenic was not linear and became saturate when concentration of arsenic reach about 3 µM (Figure 3b). The results were agreed with data described before for this E. coli DH5α (pJAMA-arsR) biosensor (Stocker, 2003). Assuming that detection limit is value equal to 3 times of standard deviations measured by blank samples, here it was seen as 0.1µM arsenic (7.5 µg/L). The sensitivity of the biosensor is adequate to identify arsenic concentration in groundwater as low as 10 µg/L, which is recommended value from WHO for arsenic criteria in drinking water.
Figure 2: Cross checking for arsenic determination by AAS (CETASD) and AFS (EAWAG) AAS and the biosensor assay were then used simultaneously at CETASD to measure arsenic levels in 194 groundwater samples that were collected in July 2004 from the Red River and Mekong delta region. Biosensor response to unknown samples was compared to a standard curve prepared in arsenic-free groundwater with the range of 0, 0.1, 0.2, 0.4, 0.8 and 1µM arsenic, standard and unknown samples always were prepared and measured at
the same batch. Of all samples two fold dilutions were measured as described at protocol (Figure 1). A comparative plot of all values gathered by AAS and biosensor showed a good correlation between both methods at Figure 4a, especially at the low concentration from 0.1 to 1µM (r2 = 0.88) at Figure 4b. Practically by AAS method, water samples with arsenic levels from < 0.1µM to < 1µM were diluted 10 time, and 100 time for higher 1µM, but o- nly two time dilution was applied for biosensor test. That could be the reason for lower response at biosensor for samples with arsenic level higher than 2µM. As show at Figure 3, the bacteria biosensor could only give the linear response at concentrations from 0 to 1µM, here with the samples contain exceed 2µM of arsenic the plot was not straight anymore and become saturated and even go down when arsenic concentration is higher than 4µM (300 µg As/L), that might be toxic levels for bacteria. Since the biosensor measures rather accurately in the lower range of arsenic concentrations (10-100 µg As/L) it has an important advantage over most other field kits at present. Assuming that the data obtained by AAS had a higher probability for
being true, the comparative false positive and false negative results obtained by the biosensor assay were calculated in Table 2 for arsenic concentrations in the range of smaller than 10, from 10 to 100 and higher than 100 µg As/L. Table 1: Some chemical compositions of groundwater at Vietnam The biosensor prediction was calculated for false negative (identifying a sample as lower than the set value, for example drinking water standard with10 µg As/L, when its true concentration by AAS is exceeding), and false positive
(identifying as exceeding the set value when it is less).
Both of these false identities are important, the false negative will mark a well as safe but actually it is not safe, this subsequently bring people to the health risk. The false positive will mark a well as not safe then should be closed for example, but actually it is safe for use, it is major social economic impact (Rahman, 2002). Among total 194 tested samples, 112 samples (57.7%) determined as safe with lower than 10 µg/L arsenic, 38 samples (19.6%) contained arsenic concentration from 10 to 100 µg/L and 44 samples (22.7%) above 100 µg/L arsenic. At level lower than 10 µg/L, 9 samples were false negative (8.0%) with arsenic levels from 10 to 19 µg/L, it means that arsenic concentrations of the false negative samples were not exceeded very much but still is the safety level of some other countries as Bangladesh (50 µg/L). With 38 samples identified in between 10 and 100 µg/L of arsenic, 5 samples (13.1%) were recorded as false negative with arsenic levels in the range of 142 - 176 µg/L and 2 samples (5.3%) were false positive. For 44 samples were determined as
contained higher than 100 µg/L of arsenic non of them was false negative and one sample (2.3%) was false positive with true value as 97 µg/L, that is very closed to 100 µg/L, this discrepancy could also happen between modern instrument tests (Kiniburgh, 2002). In summary, if all the wells were categorised as safe or not safe base on the recommended value of WHO for arsenic in drinking water (10µg/L), data recorded as 9 samples among 112 samples were false negative (8.0%) and 2 samples among 82 samples were false positive (2.4%). Table 2: False negative and false positive values of Biosensor compared with AAS In light of the horrifying high rate of false negatives with chemical field test kits of up to 68% at arsenic
concentrations in the range of 50 to 100 µg/L (Rahman, 2002), the performance of the biosensor assay is very promising. Validation with more real samples and more dilution with high arsenic contaminated samples will definitively lead to an even better idea on the accuracy of the biosensor measurements for samples with a variety of different chemical composition, but we are confident that assay using the luminescent bacterial strain E. coli DH5 (pJAMA-arsR) can be an important new tool for rapid screening of arsenic in groundwater. The test could be performed as arrays in 96 wells tray with triple measurement for each sample ensuring its accuracy. Average through put sample for 96 wells and single vial testing is 100 and 50 samples per day respectively in our lab. Likewise, similar biosensor strains selective to other chemical target compounds may herald a relatively easy and rapid tool for screening. 4. CONCLUSION