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 HNO
3
(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 r
2
=0.9). Kết quả này vượt trội hơn so với

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

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
HNO
3
(7.5M, Merck) to final concentration of 0.015M.
Water bottles were transfer to the lab, stored at 4
o
C 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

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 (Na
4
P
2
O
7.
10 H
2
O,
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

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 r
2
-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.

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

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

Our study developed a suitable protocol using the
luminescent genetically modified strain E. coli DH5
(pJAMA-arsR) for rapid screening of arsenic in
groundwater especially in iron rich media, which is
common at many high-risk areas in the world. The
biosensor showed very good accuracy at critical range of
arsenic concentration (10-100 µg/L) with r

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