ISTC Reports

Illinois Sustainable Technology Center

 
New Catalytic DNA Fluorescent  
and Colorimetric Sensors for  
On‐site and Real‐time Monitoring 
of Industrial and Drinking Water 
 
 
 
 
Yi Lu  
Department of Chemistry 
University of Illinois at Urbana‐Champaign 
 
 

 
 

RR‐114
February 2009
www.istc.illinois.edu
 RR-114

New Catalytic DNA Fluorescent  
and Colorimetric Sensors for  
On‐site and Real‐time Monitoring 
of Industrial and Drinking Water 
 
 
 
 
Yi Lu
Department of Chemistry
University of Illinois at Urbana-Champaign

February 2009

Submitted to the
Illinois Sustainable Technology Center
Institute of Natural Resource Sustainability
University of Illinois at Urbana-Champaign
www.istc.illinois.edu

The report is available on-line at:

http://www.istc.illinois.edu/info/library_docs/RR/RR-114.pdf

Printed by the Authority of the State of Illinois

Patrick J. Quinn, Governor
This report is part of ISTC’s Research Report Series (ISTC was formerly
known as WMRC, a division of IDNR). Mention of trade names or
commercial products does not constitute endorsement or recommendation
for use.
I. Acknowledgments

We wish to thank the Illinois Sustainable Technology Center, a division of the Institute of
Natural Resource Sustainability at the University of Illinois at Urbana-Champaign, for their
support of this work under grant no. HWR04187.

iii
 CONTENTS

I. Acknowledgments ……………………………..……….……………..………...…... iii

II. Tables …………………………………………….....……….…………………….... vi

III. Figures ……………………………………..………………………...………….… vii

IV. Abstract …………………………………..…………………………...………........ ix

1. Introduction ……………………………………………………………………..…… 1
i. The need for metal sensor technology for water monitoring …………………….… 1
ii. Designing metal sensors …………………..………………………………….…… 1
iii. Advantage of catalytic DNA as sensors …...…………………………………...… 2
iv. Advantages of fluorescent sensors ...…………………………………………….... 3
v. Advantages of colorimetric sensors ..…………………………………………….… 3

2. Results and discussion ………………………………………………………….……. 3

i. In vitro selection of catalytic DNA for sensing applications …………………….… 3
1) In vitro selection scheme ………………………………...…………………..... 4
2) Uranium …………………………………………………………………….…. 4
3) Arsenic ..…………………………………………...…………………………... 4
4) Other metal ions ..…………………………………………………………….... 5
ii. Design of fluorescent uranium biosensors: a catalytic beacon sensor with
parts-per-trillion sensitivity and million-fold selectivity ……………...….…….... 6
iii. Design of fluorescent copper biosensors ……………………………….………… 9
iv. Design of fluorescent mercury biosensors ……………………………….……… 11
v. On-site Pb2+ detection using a DNAzyme biosensor coupled with a portable
fluorometer ……………………………………………………………………... 14
In simulated water samples with various hardness …………………………….. 16
Real sample test; Crystal Lake water in Urbana, IL …………………………… 16
vi. Colorimetric Pb2+ sensors based on DNAzyme-assembled gold nanoparticles .... 18
vii. Towards more practical applications: simple “dipstick” tests ………………….. 19
viii. The shelf-life and operation of the sensor in a real-world setting ……..………. 20

iv
3. Summary and Outlook ………………………………………….………………….. 21

4. References ……………………………………………………………………….…... 23

v
 II. Tables

1. Reaction time and detection limits calculated from each standard curve of fluorescence
versus [Pb2+] in the simulated water samples with different hardness and Crystal Lake water
sample ……………………………………………………………………………… 18

vi
 III. Figures

1. In vitro selection protocol. See text for descriptions ……………………………….... 5

2. Design of a catalytic beacon to detect UO22+ ……..…………………………………. 7

3. Sensitivity of the catalytic beacon-based UO22+ sensor ….………………………….. 8

4. Selectivity of the catalytic beacon-based UO22+ sensor ……….…………………….. 9

5. (A) The secondary structure of the Cu2+ sensor; (B) Signal generation mechanism of the Cu2+
catalytic beacon; (C) Fluorescence spectra of the sensor before and 10 min after addition of
20 µM Cu2+; Inset: gel-based assay of the sensor DNAzyme ….……………………10

6. (A) Kinetics of fluorescence increase over background at varying Cu2+ levels. Inset:
responses at low Cu2+ levels; (B) The rate of fluorescence enhancement plotted against Cu2+
concentration. Inset: rates at the low Cu2+ region; (C) Sensor
selectivity ………………….…………………………………….……….…………. 11

7. (a) The secondary structure and modification of the Hg2+ sensor DNAzyme; (b) Fluorescence
spectra of the sensor in the absence and 8 min after addition of 0.5 µM Hg2+; (c) Schematic
presentation of the sensor design ………………………...………………….……… 12

8. Sensitivity of the Hg2+ sensor …….....…………………………………….………... 13

9. Sensor selectivity . ……………………………………………………….……….… 14

10. (A) Secondary structure of the DNAzyme; (B) Design of a catalytic beacon sensor with a
fluorophore and two quenchers .…………………………………………...……….. 15

11. Fluorescence increases at different reaction time by Pb2+ions in Millipore water ..... 15

12. Standard curves of fluorescence enhancement versus Pb2+ concentrations in simulated water
samples with different hardness (a-e) and in Crystal Lake water (f) ………….……. 17

vii
13. (A) The secondary structure of the Pb2+-specific DNAzyme; (B) In the presence of Pb2+, the
substrate is cleaved into two pieces; (C) Pb2+-directed assembly of DNAzyme-linked
nanoparticle aligned in a head-to-tail manner; (D) UV-vis spectra of disassembled (red) and
assembled (blue) gold nanoparticles; (E) The assembly state or color of DNAzyme-linked
nanoparticles in response to metal ions monitored by a spectrophotometer, or on a TLC plate
(F) ………………………………..…………………………………………………. 19

14. Dipstick detection of toxic metal ions such as Pb2+ based on catalytic DNA and gold
nanoparticles …………………………………..……………………………………. 20

15. (A) Stability study of the catalytic DNA-based lead sensor after drying at different
temperatures. Stability at 45 oC (B) and 80 oC (C). (D) Stability of DNA attached to gold
nanoparticles .………………………………………… ……………………………. 21

viii
 IV. Abstract

We have developed new fluorescent and colorimetric sensor technologies for on-site, real-time
detection and quantification of toxic metal ions such as lead, mercury and uranium in industrial
and drinking waters. We used a combinatorial biology method called in vitro selection to obtain
catalytic DNA with high specificity and selectivity for the metal ions. By labeling the DNA with
either fluorophore/quencher pairs or gold nanoparticles, we have transformed the catalytic DNA
into a highly sensitive and selective fluorescent or colorimetric biosensor, respectively. The
presence of metal ions causes the catalytic DNA to cleave, resulting in either a dramatic increase
of fluorescent signals or a distinctive change of colors. The sensors are highly sensitive (with
detection limit as low as 11 ppt), and selective (with selectivity of over millions fold). The
catalytic DNA fluorescent biosensors make it possible to analyze metal ions using simple
portable fluorometers, and the catalytic DNA colorimetric biosensors can eliminate equipment
altogether. This is possible because the toxic metal ions can be detected through simple color
changes, just like pH paper.

ix
1. Introduction

i. The need for metal sensor technology for water monitoring. According to publications
from the Illinois Department of Public Health (IDPH),1 lead poisoning is the No. 1
environmental illness effecting children. In Illinois, ~25,000 children per year are identified with
elevated blood lead levels. One of the main sources of lead in the environment is lead-based
paint. About 75% of homes built before 1978 contain some leaded paint. In addition, IDPH has
issued special mercury advisory, cautioning Illinois residents about the danger of eating certain
fish species containing mercury in selected Illinois rivers and lakes. Finally, a recent report
indicated that up to one-third of private wells in selected counties surrounding the Mahomet
aquifer have elevated levels of arsenic.2 This aquifer is the main water source for central Illinois.
Therefore, it is important to identify and quantify these and other toxic metal ions in our
environment. New materials and technology3 have been developed to reduce or eliminate those
type of ions. Assessing the success of the materials and technology in removing those ions from
water requires new analytical techniques.

Analytical techniques, such as atomic absorption spectrometry,4-6 inductively coupled plasma

mass spectrometry,7-9 anodic stripping voltammetry,10, 11 capillary electrophoresis,12 x-ray
fluorescence spectrometry13-15 and microprobes,16-23 have been routinely used for metal ion
analysis with high sensitivity (often ≤ ppb level). Many of them can quantify many metal ions
simultaneously. However, it is generally believed that most of the above techniques require
sophisticated equipment, sample pretreatments, or skilled operators, making it difficult to do on-
site, real-time monitoring of metal ions.24-26 Due to the toxicity these metal ions may pose to
operators, remote sensing devices are desirable.27-29 While important progress has been made to
miniaturize many of the above analytical instruments,30-34 design of sensitive and selective metal
sensors using either cost-effective and portable equipment or no equipment at all provides an
effective alternative means of achieving the goal of industrial and drinking water monitoring.

ii. Designing metal sensors. Fluorosensors based on fluorescently-labeled organic

chelators,24, 26, 35, 36 proteins37-39 or peptides40-43 have emerged as powerful tools toward achieving
the above goal.44 While remarkable progress has been made in developing fluorosensors for
2+ 2+
some metal ions such as Ca 24, 38 and Zn ,39-41 designing and synthesizing sensitive and
selective metal ion fluorosensors remains a significant challenge. Perhaps the biggest challenge
in fluorosensor research is the design and synthesis of a sensor capable of specific and strong
metal-binding. Since knowledge about the construction of metal-binding sites is limited,
searching for sensors in a combinatorial way is of significant value. For example, even though

1
colorimetric sensors can allow onsite, real-time qualitative or semi-quantitative detection without
complicated analytical instruments, the design of such sensors is not very advanced.45-48 In this
14 15
regard, in vitro selection of DNA/RNA from a library of 10 -10 random DNA/RNA sequences
offers considerable opportunity.49, 50 Compared with combinatorial searches of chemo- and
peptidyl-sensors, in vitro selection of DNA/RNA is capable of sampling a larger pool of
sequences, amplifying the desired sequences by the polymerase chain reaction (PCR), and
introducing mutations to improve performance by mutagenic PCR. For example, the in vitro
selection method has been used to obtain DNA/RNA aptamers51, 52 and aptazymes53, 54 that are
responsive to small organic molecules. Similarly, catalytic DNA/RNAs that are highly specific
2+ 2+ 2+
for Pb ,49, 55 Cu 56, 57 and Zn 58, 59 have been obtained. These results set the stage for the
utilization of catalytic DNAs as metal ion sensors.

iii. Advantage of Catalytic DNA as sensors. Long considered as simply a genetic material,
DNA was shown in 1994,49 through in vitro selection,49, 60-63 to carry out catalytic functions, and
thus became the newest member of the enzyme family (after proteins and RNA). Since then, the
DNA molecules (called catalytic DNA in this paper, also called DNA enzymes, DNAzymes or
deoxyribozymes elsewhere) have been shown to catalyze many of the same reactions as catalytic
RNA (ribozymes) or protein enzymes.64-67 Several features make catalytic DNA an excellent
choice for sensing applications. The first and perhaps biggest advantage of choosing catalytic
DNAs is that they can be subjected to in vitro selection. When compared to other combinatorial
methods based on organic chelators or peptides, in vitro selection can sample a larger pool of
different molecules (up to 100 trillion), amplify the desired sequences by the polymerase chain
reaction (PCR), and introduce mutations to improve performance by mutagenic PCR. Second,
the in vitro selection can be carried out in short time and with limited cost (1-2 days and a few
dollars per round of selection). Third, the synthesis of DNA is easier, and therefore less costly
than the synthesis of RNA. Under physiological conditions, DNA is nearly 1,000-fold more
stable to hydrolysis than proteins and nearly 100,000-fold more stable than RNA.66 As seen from
a recent crystal structure,68 catalytic DNAs usually form compact globular-shaped proteins and
are therefore not easily recognized by endo- or exonucleases, and thus are likely more resistant to
nuclease attack than single or even double-stranded DNA/RNA.69 Fourth, unlike proteins, most
catalytic DNA can be denatured and renatured many times without losing binding ability or
activity. They can be used and stored under rather harsh conditions. Fifth, DNA is adaptable to
fiber optic and microarray technology,70, 71 which is important for on-site or remote sensing of
multiple metal ions simultaneously.

2
 iv. Advantages of fluorescent sensors. We intend to convert the in vitro selected catalytic
DNAs into fluorescent sensors. Fluorescence provides significant signal amplification because a
single fluorophore can absorb and emit many photons, which leads to strong signals even at very
low concentrations of fluorescent probe or analyte. In addition, the fluorescence time-scale is fast
enough to allow real-time monitoring of concentration fluctuations. Fluorescent properties only
respond to changes related to the fluorophore and can be highly selective. Furthermore, portable
fluorometers for use in the field are available.72-74 Fluorescence detection is also compatible with
fiber-optic technology and well suited for remote sensing applications.27-29, 75-81 As demonstrated
recently,82 there are three additional advantages of catalytic DNA fluorescent sensor systems.
The metal sensing is achieved by both metal-binding and catalytic activity, allowing signal
amplification through catalytic turnover. The fluorophores can be placed remotely from the
binding and cleavage sites so that binding and sensing do not interfere with each other and can
be optimized independently. Finally, the effective placement of the fluorophores can be
accomplished with little knowledge of the three dimensional structure of the system.

v. Advantages of colorimetric sensors. While fluorescent sensors offer many advantages,

a simple colorimetric detection method could eliminate or minimize most costs associated with
the instrumentation and operation of fluorescence detection and thus can make on-site, real-time
detection even easier. The colorimetric sensors described here combine cutting edge technologies
in biology (in vitro selection of catalytic DNA) and nanotechnology (gold nanoparticles). Test
results showed that the sensitivity and selectivity of this method rivals, and in many cases
exceeds, other methods such as those based on fluorescence.

The results of our research demonstrate that the use of catalytic DNA for metal detection is
an effective way to identify metal poisoning problems.

2. Results and discussion

i. In vitro selection of catalytic DNA for sensing applications. Catalytic DNA molecules
(also called DNAzymes) were first isolated by Breaker and Joyce in 199449. A combinatorial
biology technique called in vitro selection has been used to isolate DNAzymes that can perform
a number of different catalytic functions such as RNA cleavage49, ligation56, 83, 84,
phosphorylation85 and porphyrin metallation86. In a typical in vitro selection process, we start
with a random DNA library containing 1013 – 1016 sequences. The sequences that exhibit a
desired function are separated by techniques such as column chromatography, gel based
separation and more recently capillary electrophoresis87. These active sequences are then
amplified and subjected to additional rounds of selection, often increasing the selection

3
stringency. During each round, ‘winner’ sequences are amplified and the non active sequences
are removed. The process is repeated to obtain the optimally active sequence. We are interested
in obtaining RNA cleaving DNAzymes that cleave a single ribo-linkage embedded in a DNA
sequence, only in the presence of a particular metal ion of interest. This forms the basis of
DNAzyme based metal ion sensors.

1) In vitro selection scheme - In vitro selection has been carried out using a protocol shown in
Figure 1. The template containing a 50 nucleotide random region is extended and then amplified
to obtain the random pool containing a single riboadenosine cleavage site. In each round of
selection, the DNA pool dissolved in selection buffer is incubated with the metal ion at a desired
concentration. Some molecules in the initial pool will be catalytically active and will be able to
catalyze the cleavage reaction at the riboadenosine site. The cleavage product is separated from
the uncleaved pool by gel electrophoresis and extended to the full length of the random pool and
amplified by PCR. The selection pressure is progressively increased so as to obtain the most
efficient sequences that can catalyze the cleavage reaction in the minimum amount of time and
metal ion concentration.

The selection scheme is presented in Figure 1. A total of three primers were used in two
polymerase chain reactions (PCR). In the first PCR, P3 and P4 were used to generate a full-
length pool; rA was introduced through P3, P4 contained a PEG spacer (Spacer-18, denoted as a
green dot) that was incorporated into the negative strand without rA. The purpose of the spacer
was to stop the PCR extension. As a result, two strands of unequal lengths were produced, and
the positive strand was purified by denaturing polyacrylamide gel electrophoresis (PAGE). In the
second PCR, based on the cleaved products, P2 and P4 were used to generate the selection pool
for the next round of selection.

2) Uranium - UO22+ was added to search for DNAzymes that can perform the self-cleavage
reaction and the cleaved products were separated by PAGE to seed the next round of selection.
Initially, 1 mM of UO22+ with 5 hour reaction time was used, which gradually decreased to 0.1
mM and 15 minutes in round 11. The round 10 pool was cloned and 86 sequences were obtained.
After performing activity assays on individual clones, clone 39 was chosen for uranium sensing.
After truncation and rational design of substrate binding sequences, a trans-cleavage DNAzyme
was constructed and converted into a fluorescent sensor with a detection limit of 45 pM (see
section 2-ii for details).

3) Arsenic - Using a similar protocol as described above, we had carried out in vitro selection of
As3+ and As5+-specific DNAzymes. Because As3+ and As5+ are in the form of oxyanions, it has

4
been difficult to obtain negatively-charged DNAzymes that have specific activity toward those
metal ions. After three attempts of in vitro selection at 10 rounds each, we obtained DNAzymes
with no detectable activity in the presence of As3+ or As5+. We hypothesized that the DNAzyme
templates used in the early selection tend to favor metal ions such as Pb2+. Based on the work of
Cruz et al.,88 a new template that is less biased toward Pb2+ was chosen, and used for in vitro
selection. The 8th round of in vitro selection of As3+ and As5+-specific DNAzyme has been
completed using a newly designed template.

4) Other metal ions - Protocols for in vitro selection of Pb2+49, 55 and Cu2+56, 57 -specific
DNAzymes have been reported in the literatures previously. Hg-specific allosterically controlled
DNAzymes was modified from in vitro selected UO22+-specific DNAzymes (see section 2-iv for
details).
random region
N50
Template

rA P3

Template directed
extension

rA

rA

P4 STOP
PCR amplification
of initial pool

rA
STOP

Gel purification
PCR extension
of random pool
and amplification

STOP

rA
STOP

P2
rA M2+
PCR extension of Cleavage
cleavage product
rA
(cleavage products)

STOP
Gel purification of
cleavage product
rA
(uncleaved)

Figure 1. In vitro selection protocol. See text for descriptions.

5
 ii. Design of fluorescent uranium biosensors: a catalytic beacon sensor with parts-per-
trillion sensitivity and million-fold selectivity. We obtained a UO22+-specific DNAzyme and
demonstrated a highly sensitive and selective UO22+ sensor based on the DNAzyme. A catalytic
beacon sensor for uranyl (UO22+) consists of a DNA enzyme strand with a 3′ quencher, and a
DNA substrate with a ribonucleotide adenosine (rA) in the middle and a fluorophore and a
quencher at the 5′ and 3′-ends, respectively (Figure 2). The presence of UO22+ causes catalytic
cleavage of the DNA substrate strand at the rA position and release of the fluorophore and thus a
dramatic increase in fluorescence intensity. As shown in Figure 3A, the rate of fluorescence
enhancement was faster in higher concentrations of UO22+. F/F0 in the y-axis is the fluorescence
at a certain time over background fluorescence before addition of uranium, which thus represents
the fold of fluorescence increase. The fold of fluorescence increase at different uranium
concentrations was plotted in Figure 3B. The sensor’s performance was evaluated by conducting
linear regression over the lower concentration portion of curve in Figure 3B: detection limit of
11 parts-per-trillion (45 pM), and dynamic range up to 400 nM. This sensor rivals the most
sensitive analytical instruments for uranium detection. Its application in detecting uranium in
contaminated soil samples is also demonstrated.95 This work shows those simple, cost-effective,
and portable metal sensors have similar sensitivity and selectivity to much more expensive and
sophisticated analytical instruments. With wide availability of portable fluorometers, such a
highly sensitive and selective UO22+ sensor will find wide applications in on-site and real-time
environmental monitoring. Successes with the uranium sensor demonstrated that the DNAzyme
sensing platform has an enormous potential for detection and quantification of many metal ions.

6
 B

Figure 2. Design of a catalytic beacon to detect UO22+. (A) The secondary structure of an in
vitro selected DNAzyme specific to UO22+, which contains a substrate (39S) and an enzyme
(39E). (B) Design of a catalytic beacon with a fluorophore and two quenchers. Cleavage of the
substrate in the presence of UO22+ enhances the fluorescence. (C) Fluorescence signal in the
absence and in the presence of 400 nM UO22+ after 10 minutes. The UO22+ sensor was in pH 5.5
MES buffer with 300 mM NaCl.

7
 Figure 3. Sensitivity of the catalytic beacon-based UO22+ sensor. (A) Kinetics of fluorescence
increase over background fluorescence at varying UO22+ concentrations. The DNAzyme sensor
concentration was 60 nM, and the buffer contained 50 mM MES (2-(N-morpholino)ethanesulfonic
acid) (pH 5.5) and 300 mM NaNO3. (Inset) Sensor responses to low concentrations of UO22+. F/F0
in the y-axis is the fluorescence at a certain time over background fluorescence before addition of
uranium, which thus represents the fold of fluorescence increase. (B) Plot of the initial rate of
fluorescence enhancement (Vfluo, fold of fluorescence increase per minute) vs. UO22+ concentration.
(Inset) Low UO22+ concentration range with linear responses.

To test the selectivity of the sensor for UO22+ vs. other metal ions, the sensor response to 19
competing metal salts at concentrations of 10 µM, 200 µM, and 1 mM was tested. Most metals
induced little fluorescence change, although some metals such as Cu2+, Fe2+, Fe3+, Hg2+, and
Tb3+ induced strong quenching to FAM (5-Carboxyfluorescein). Similarly, the rate of
fluorescence change was calculated. As shown in Figure 4, at all three concentrations, none of
the metals [except Th(IV)] showed a response higher than that of 1 nM UO22+. Some Th(IV)-
dependent fluorescence increase was observed. However, gel-based assays showed no Th(IV)-
dependent cleavage even with 1 mM Th(IV) (see supplemental information in ref. 95). Therefore,
Th(IV) interference is likely to be due to metal/fluorophore interactions, which can be removed

8
by using other fluorophores. Overall, the sensor has over one million-fold selectivity for UO22+
over any other metal ions.

Figure 4. Selectivity of the catalytic beacon-based UO22+ sensor. Sensor responses to all competing metal ions at
three concentrations (10 µM, 200 µM, and 1 mM) were tested.

iii. Design of fluorescent copper biosensors. Copper is a widely used metal that can enter
the environment through various routes. At low concentration, copper is an essential nutrient to
human beings. However, exposure to high level of copper even for a short period of time can
cause gastrointestinal disturbance. Long term exposure causes liver or kidney damage.89
Therefore, regulations for copper in drinking water have been established. For example, the US
Environmental Protection Agency (EPA) defines the limit of copper in drinking water to be 1.3
ppm (~20 µM).

We chose a Cu2+-dependent DNA-cleaving DNAzyme reported by Breaker et al as a basis

for the sensor design.90-92 Based on the original DNAzyme sequence, we designed a Cu2+ sensor
as shown in Figure 5A. The sensor contained two DNA strands that can form a complex. The
substrate strand (in black) was labeled with a FAM fluorophore (6-carboxyfluorescein) on the 3'-
end, and a quencher (Iowa Black FQ) on the 5'-end. The enzyme strand (in blue) contained a 5'-
quencher. A dual-quencher approach was employed to suppress background fluorescence.93 The
substrate and enzyme associate through two base pairing regions. The 5' region of the enzyme
binds the substrate via Watson-Crick base pairs; while the 3' region binds through formation of a
DNA triplex. Initially, the FAM emission was quenched by the nearby quenchers. In the
presence of Cu2+, the substrate was cleaved at the RNA site (the guanine in red). We
hypothesized that the cleaved pieces were released due to decreased affinities to the enzyme,
leading to increased distance between the FAM and the quenchers and increased fluorescence
(Figure 5B). This hypothesis was supported by the observation that the FAM emission increased

9
by ~13-fold after addition of 20 µM Cu2+ (Figure 5C). Such a signal generation method was
termed “catalytic beacon” because of the involvement of catalytic reactions.94-96 Gel-based
assays were carried out to confirm that the observed fluorescence increase was due to DNAzyme
cleavage (inset of Figure 5C). Lane 1 contained the substrate alone incubated with Cu2+. Lane 2
had the DNAzyme complex but no Cu2+. Cleavage was observed only after addition of Cu2+ to
the DNAzyme complex and the fraction of cleaved substrate increased over time (lanes 3-5),
which supported the proposed mechanism in Figure 5B. In all the reactions, 50 µM of ascorbate
was included. Although this DNAzyme has been shown to work in the absence of ascorbate,90-92
the reaction rates were significantly enhanced by ascorbate. Ascorbate was also useful in this
system to suppress quenching of FAM by Cu2+. For example, FAM quenching was <15% with
50 µM Cu2+.

Figure 5. (A) The secondary structure of the Cu2+ sensor. F and Q denote fluorophore and quencher, respectively.
The cleavage site is indicated by an arrow. (B) Signal generation mechanism of the Cu2+ catalytic beacon. (C)
Fluorescence spectra of the sensor before and 10 min after addition of 20 µM Cu2+. Inset: gel-based assay of the
sensor DNAzyme. Lane 1 is substrate plus Cu2+, Lane 2 is substrate plus enzyme, Lanes 3 to 5 are 5, 10, and 25
min after addition of Cu2+, respectively.

To test the sensitivity of the Cu2+ sensor, the kinetics of fluorescence increase at 520 nm in
the presence of varying concentrations of Cu2+ were recorded on a fluorometer. As shown in
Figure 6A, fluorescence enhancement rates were higher with increasing levels of Cu2+. The rates
in the time window of 2 to 4 minutes were plotted in Figure 6B. A detection limit of 35 nM (2.3
ppb) was determined, which represents one of the most sensitive Cu2+ sensors.97 The sensor has a
dynamic range up to 20 µM. This is very useful for detecting Cu2+ in drinking water because the
US EPA has defined a maximum contamination level of 20 µM. In addition to being highly
sensitive and “light-up”, the sensor response was also fast, so quantitative results can be obtained
within several minutes.

10
 To test selectivity of the sensor, 16 competing metal ions were assayed at three different
concentrations: 1 mM, 100 µM, and 10 µM. The assay was performed in a 96-well plate and
emission intensities at 12-min after addition of metal ions were compared. As shown in Figure
6C, besides Cu2+, only the spots with 1 mM Fe2+ and 1 mM UO22+ lit up and the intensities were
lower than that with 0.5 µM of Cu2+. Therefore, the sensor selectivity for Cu2+ was at least
2,000-fold higher than these two metals, and >10,000-fold higher than any other metal ions. Fe2+
is known to cleave DNA through Fenton’s chemistry.98 For testing environmental samples, such
as detection of Cu2+ in drinking water, Fe2+ is unlikely to interfere due to the oxidative
environment. UO22+ is also unlikely to present in millimolar concentration in drinking water.

(µM)

Figure 6. (A) Kinetics of fluorescence increase over background at varying Cu2+ levels. The arrow indicates the
point of Cu2+ addition. Inset: responses at low Cu2+ levels. (B) The rate of fluorescence enhancement plotted against
Cu2+ concentration. Inset: rates at the low Cu2+ region. (C) Sensor selectivity. The buffer contained 1.5 M NaCl, 50
mM HEPES, pH 7.0, and 50 µM ascorbate. Cu2+ concentrations were labeled on the left side of each well while
others were on the right end. All concentrations are in µM.

iv. Design of fluorescent mercury biosensors. Mercury ions are known to bind in between
T-T mismatches to stabilize such mismatches.99 Based on this knowledge, the catalytic DNA
sensor for Hg2+ detection is shown in Figure 7a. This catalytic DNA was built on the basis of the
uranium catalytic DNA shown in Figure 2A of this report. A fluorophore (FAM) was labeled on

11
the 5'-end of the substrate, a quencher was labeled on the 3'-end of the enzyme, and an additional
quencher was labeled on the 3'-end of the substrate. Both quenchers were black hole quenchers.
The DNAzyme was mixed with UO22+ to become an Hg2+ sensor (Figure 7b). In the absence of
Hg2+, the DNAzyme was incapable of binding UO22+ because the active secondary structure
cannot form. Addition of Hg2+ quickly restored the stem loop structure and activated the
DNAzyme to cleave the substrate, releasing the fluorophore-labeled piece and giving increased
fluorescence. The fluorescence spectra of the sensor before and 8 min after addition of 500 nM
Hg2+ is shown in Figure 7c, and ~50-fold increase in the 520 nm peak was observed. This level
of fluorescence increase is the highest in functional nucleic acid based sensors for metal ions.

Figure 7. (a) The secondary structure and modification of the Hg2+ sensor DNAzyme. (b) Fluorescence spectra of
the sensor in the absence and 8 min after addition of 0.5 µM Hg2+. (c) Schematic presentation of the sensor design.

Given the very high fluorescence enhancement, the sensor performance was further tested.
First, the sensor was titrated with varying concentrations of Hg2+ and the kinetics of fluorescence
enhancement at 520 nm was followed. As shown in Figure 8a, higher concentrations of Hg2+
produced higher rates of emission enhancement. All the kinetic traces showed a roughly linear
increase in 1-2 min after addition of Hg2+. Therefore, the slope of fluorescence increase in this
time window was calculated to quantify Hg2+ concentrations (Figure 8b). The Hg2+ dependent
response had a sigmoid shape and was fit to a Hill plot with a Hill coefficient of 2.1. This result
suggests that Hg2+ binding to the sensor DNAzyme is a cooperative process. Although the
DNAzyme has five Hg2+ binding sites, the DNAzyme is stable enough in the time window to
cleave its substrate after binding ~2 Hg2+ ions. The detection limit was determined to be 2.4 nM
(~0.5 ppb) based on 3/slope (inset of Figure 8b), which is a ~16-fold improvement compared to

12
the previous oligonucleotide-folding based sensor.99 Based on our results, among all the reported
Hg2+ sensors made from small molecules, oligonucleotides, peptides, and proteins, this catalytic
beacon has the best detection limit. The US Environment Protection Agency (EPA) defined the
toxic level of Hg2+ in drinking water to be 2 parts-per-billion or 10 nM, which can be covered by
the sensor.

Figure 8. Sensitivity of the Hg2+ sensor. (a) Kinetics of fluorescence increase in the presence of varying
concentrations of Hg2+. All the reactions were carried out at 24 ºC in 10 mM MES, pH 5.5, 300 mM NaNO3 with
100 nM DNAzyme sensor and 1 µM UO22+. (b) Hg2+ dependent fluorescence increase rate. Rates were calculated in
the time window of 1-2 min in (a). Inset: responses at low Hg2+ concentrations. The y-axis is the fluorescence counts
increase per second.

To test selectivity, the sensor responses in the presence of 13 competing metal ions were
assayed. These metal ions included Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Pb2+,
UO22+, and Tb3+. Each metal ion was tested at three concentrations (1, 20, and 1000 µM). None
of the metal ions gave responses higher than half of that produced by 20 nM Hg2+ (Figure 9), and
the selectivity was determined to be at least 100,000-fold higher for Hg2+ over any other metal
ions (10 nM Hg2+ versus 1 mM other metal ions).

13
Figure 9. Sensor selectivity. All competing metal ions were tested at 1, 20, and 1000 µM. For comparison, sensor
responses to 20, 100, and 500 nM of Hg2+ were also presented.

v. On-site Pb2+ detection using a DNAzyme biosensor coupled with a portable

fluorometer. After demonstrating individual fluorescent sensors for heavy metal detection, the
use of the Pb2+ sensor for detecting Pb2+ in drinking water samples was carried out.

The secondary structure of the DNAzyme is shown in Figure 10. For the Pb2+ sensor, the
substrate strand was labeled with a fluorophore (Fluorescein or Alexa fluro 488) at the 5‫׳‬-end and
a quencher (Dabcyl) at the 3‫׳‬-end (FQ-17S). The enzyme strand was labeled with a quencher
(Dabcyl) at the 3‫׳‬-end (Q-17E). The Pb biosensor was 2 μM DNAzyme in 50 mM Tris-HCl
(pH7.3) and 10 mM NaCl. Fluorescence enhancements were measured by using a portable
fluorometer, PicofluorTM (Turner Designs). Measurements were made after the addition of 5 μL
of 2 μM DNAzyme sensor into the 95 μL of Pb2+ solutions buffered with 50 mM Tris-HCl
(pH7.3) + 30 mM NaCl. Reaction time varied depending on the samples’ ionic strength.

14
 A
17S
3' G T A G A G A A G G rA T A T C A C T C A 5'
5' C A T C T C T T C T A T A GT GA GT 3'
17E C A
C A
A G G G
G T C
G C
C
B FQ-17S
Q F
Q
Q-17E

2+
M =Pb

Q
Q
Q-17E M

Figure 10. (A) Secondary structure of the DNAzyme. (B) Design of a catalytic beacon sensor with a fluorophore
and two quenchers. The substrate strand, 17S contains a RNA base, rA, at the cleavage site denoted with an arrow in
(A). It was labeled with a fluorophore, either fluorescein or Alex fluro 488, at the 5‫׳‬-end and with a Dabcyl quencher
at the 3‫׳‬-end. The enzyme strand, 17E was labeled with a Dabcyl quencher at the 3‫׳‬-end. Cleavage of the substrate in
the presence of Pb2+ enhances the fluorescence.

5e+4
Fluorescence (a.u.)

4e+4

3e+4

2e+4 10 min (19 nM)

20 min (16 nM)
1e+4 30 min (10 nM)
60 min (1.8 nM)
0
0 100 200 300 400 500
[Pb2+] (nM)

Figure 11. Fluorescence increases at different reaction time by Pb2+ions in Millipore water. Reactions were started
by adding 5 μL of 2 μM DNAzyme into 95 μL of Pb2+ in reaction buffer. The DNAzyme was annealed in 50 mM
Tris-HCl(pH7.3) + 10 mM NaCl and the reaction buffer contained 50 mM Tris-HCl (pH7.3) + 30 mM NaCl. Each
data point represents three replicates. The solid lines are linear regressions at each reaction time with all 7 points for
10 and 20 min, with first 6 points for 30 min, and with first 5 points for 60 min. Detection limits are indicated inside
the brackets after each reaction time.

15
 Figure 11 shows sensor response in Millipore water. At 10 minutes reaction time, the
fluorescence showed linear increases over the entire region of 0-500 nM of Pb2+ with detection
limit of ~ 20 nM. As the reaction time increased, the curves became steeper with lower detection
limits, but the linearity was lost due to the saturated reaction time with high Pb2+ concentrations.
For each reaction time, 50 nM of Pb2+ could be easily distinguished from the background. It was
possible to detect 10 nM of Pb2+ after one hour of reaction time.

In simulated water samples with various hardness. Real world water sources contain various
kinds of materials including minerals. It has been know that Na+, K+, Ca2+, and Mg2+ are
abundant in several hundreds micromolar ranges in most real world water sources while less than
1 μM of heavy metal ions are present.103 It was shown that the DNAzyme does not have activity
in the presence of millimolar monovalent metal ions and has 1000-fold selectivity to Pb2+ over
Ca2+ and Mg2. However, it is most likely that real world water sources will contain more than
1000-fold excess of Ca2+ and Mg2+. Thus the DNAzyme’s performance was examined in the
presence of highly concentrated Ca2+ and Mg2+.

Simulated water samples of various hardness were prepared by mixing MgCl2 and CaCl2 ions
in 1:1 molar ratio and were spiked with different concentrations of Pb(OAc)2. Increasing
hardness induced faster increase in fluorescence due to the reactions not only by Pb2+ but also by
Ca2+ and Mg2+. Measuring fluorescence at short reaction time made it possible to distinguish
[Pb2+] in the low concentration region of 0–500 nM in the presence of the interferences by Ca2+
and Mg2+. Standard curves of fluorescence enhancements versus [Pb2+] in the simulated water
samples were obtained by reading the fluorescence at different reaction times (Figure 12).
Fluorescence values were background-corrected (fluorescence at 0 nM Pb2+ in each hardness was
used as the background signal). Linear curves were obtained in the fluorescence enhancement in
the range of 0 to 500 nM Pb2+. Detection limits were calculated from the slopes of the curves and
the standard deviations of the points at 0 nM of [Pb2+] (Table 1). All cases except hardness 120
mg/L have detection limits lower than the EPA standard, 72 nM, and they were reproducible. For
the case of hardness 120 mg/L, the linearity was poor and it was not easy to reproduce the same
trend. These results show that it is possible to detect Pb2+ in the moderately hard (H 60 mg/L)
water.

Real sample test; Crystal Lake water in Urbana, IL. In order to test the DNAzyme Pb2+
biosensor using a real water sample, we tested water from Crystal Lake in Urbana, IL. The water
was filtered with a 0.2 μM syringe filter and analyzed with ICP-MS. The water contained 160
pM Pb2+, 100 μM Ca2+, and 44.3 μM Mg2+, which corresponds to a hardness of 14.43 mg/L.
Since the Pb2+ concentration in the water is much lower than the level of concern and well below

16
the sensor detection limit, the water was spiked with Pb(OAc)2 for the experiments. Fluorescence
was measured at 5 minutes of reaction time. Linear fluorescence increase was shown in the range
of 0-500 nM Pb2+ with a detection limit of 44.2 nM (Figure 12f). This result implies that the
DNAzyme biosensor can detect Pb2+ in real world water sources that contain numerous minerals
and organics.
200 250 250
(a) H 0 (b) H15
H 15 (c) H 30
Fluorescence (a.u.)

Fluorescence (a.u.)
5 min

Fluorescence (a.u.)
150
10 min 200 5 min 200

150 150
100
100 100
50
50 50
y = -7.441 + 0.341 x y = -6.180 + 0.449 x y = 6.779 + 0.466 x
2 2
0 (R = 0.969) (R2 = 0.974) (R = 0.940)
0 0

0 100 200 300 400 500 0 100 200 300 400 500 0 100 200 300 400 500
2+
[Pb ] (nM) 2+
[Pb2+] (nM)
[Pb ] (nM)
250 200 200
(d) H 60 (e) H 120 (f) Crystal
Crystal lake,
Lake,Urbana,
Urbana,IL.IL.
5 min

Fluorescence (a.u.)
Fluorescence (a.u.)
Fluorescence (a.u.)

200 3 min 2 min 150

150

150
100 100
100
50 50
50 y = 3.233 + 0.301 x
y = -3.215 + 0.332 x y = -6.716 + 0.226 x 2
2 (R = 0.968)
0 (R = 0.892) 0 (R2 = 0.872) 0

0 100 200 300 400 500 0 100 200 300 400 500 0 100 200 300 400 500
[Pb2+] (nM) [Pb2+] (nM) [Pb2+] (nM)

Figure 12. Standard curves of fluorescence enhancement versus Pb2+ concentrations in simulated water samples
with different hardness (a-e) and in Crystal Lake water (f). Fluorescence was measured with a portable fluorometer,
PicofluorTM (Turner Designs). The Fluorescence intensities were background (0 Pb2+ point)-subtracted. The solid
lines show the linear regression of the data. Reaction times were 10, 5, 5, 3, 2, and 5 minutes for the hardness values
of 0, 15, 30, 60, 120 mg/L, and Crystal Lake water, respectively.

17
 Table 1. Reaction time and detection limits calculated from each standard curve of fluorescence versus
2+
[Pb ] in the simulated water samples with different hardness and Crystal Lake water sample.

Hardness (mg/L) Reaction Time (min) DL (nM)

H0 10 27.3

H 15 5 34.8

H 30 5 53.8

H 60 3 67.1

H 120 2 164

Crystal lake water 5 44.2

vi. Colorimetric Pb2+ sensors based on DNAzyme-assembled gold nanoparticles. After

demonstrating fluorescent sensors for metal detection, we aimed to further simplify the detection
system to design colorimetric sensors. We employed a Pb2+-specific RNA-cleaving DNAzyme
(Figure 13A) to direct the assembly state of gold nanoparticles in response to Pb2+.100 The Pb2+-
specific 8-17 DNAzyme was chosen as a model DNAzyme for metal sensor design. In the
presence of Pb2+, the enzyme cleaves the substrate into two pieces (Figure 13B). To allow the
DNAzyme to bind DNA-functionalized gold nanoparticles, the substrate strand was extended on
both ends and the extended substrate was named SubAu (Figure 13C).100 The nanoparticles were
aligned in a head-to-tail manner so that only one set of nanoparticles (5'DNAAu) was used. The
nanoparticles were pre-assembled by the DNAzyme to assure an optimal ratio between the
DNAzyme and nanoparticles. The assembled nanoparticle aggregates can be used as colorimetric
sensors for Pb2+ detection. To detect Pb2+, the sensor was heated to 50 ºC to fully disassemble the
aggregates. In the subsequent slow cooling process to room temperature (annealing), Pb2+ can
direct the color of the system. If Pb2+ is present, the substrate is cleaved by the enzyme and
assembly is inhibited, resulting in a red color. Otherwise, nanoparticles are re-assembled by the
DNAzyme to form aggregated structures, accompanied by a red-to-blue color change due to
surface plasmon coupling. Upon aggregation, the 522 nm plasmon peak decreases while the
extinction in the 700 nm region increases (Figure 13D), with the extinction ratio at 522 nm over

18
700 nm used to quantify the nanoparticle assembly state. A higher ratio is associated with
dispersed particles of red color, while a lower ratio is associated with aggregated particles of blue
color. With increasing concentrations of Pb2+, the extinction ratio increases, suggesting the
nanoparticles are in the disassembled state (Figure 13E). The detection limit was determined to
be 100 nM. The color change was also conveniently observed by spotting the nanoparticle
solution on a TLC plate (Figure 13F). A color progression from blue/purple to red can be
observed with increasing Pb2+ concentrations, while competing metal ions resulted in only a
background blue/purple color.

Figure. 13. (A) The secondary structure of the Pb2+-specific DNAzyme. (B) In the presence of Pb2+, the substrate is
cleaved into two pieces. (C) Pb2+-directed assembly of DNAzyme-linked nanoparticle aligned in a head-to-tail
manner. (D) UV-vis spectra of disassembled (red) and assembled (blue) gold nanoparticles. (E) The extinction ratio
increases as the Pb2+ concentration increases. The detection limit of the system could be modulated by varying the
ratio of the active enzyme (17E) and an inactived version of the sensor (17Ec) so that the 17E was 100% or only 5%
of the DNA in solution. (F) The assembly state or color of DNAzyme-linked nanoparticles in response to metal
ions monitored by a spectrophotometer or on a TLC plate.

vii. Towards more Practical Applications: Simple “Dipstick” Tests. Although

nanoparticle-based colorimetric sensors can eliminate the use of analytical instruments for
detection, there is one limitation that prevents their practical application. The handling of

19
solutions, such as the transfer of microliter volumes of sensors and their subsequent mixing with
target solutions, makes the sensors difficult for inexperienced people to use. One of the most
useful methods to convert antibody-based assays to user-friendly test kits is lateral flow
technology. A well-known example is the commercially available pregnancy test kit. Despite
wide applications in antibody assays, nucleic acid-based lateral flow devices have been
demonstrated only for DNA detection.101 We pursued the feasibility of using lateral flow devices
to design DNAzyme-based sensors that can be used as simple dip sticks. Figure 14 shows the
design for a dipstick Pb2+ sensing device. The test strip is assembled by placing a carboxy ester
membrane, glass fiber conjugate pad and adsorbent pad (Millipore Assembly kit) on a plastic
adhesive backing. The chimeric substrate has a biotin moiety on the 3' and a thiol moiety on the
5' end. There are 2 capture areas on the membrane, called the control zone (with streptavidin)
and the test zone (with DNA complementary to the 5' cleavage product). When the pad is dipped
in a flow buffer, the gold nanoparticles conjugated to the catalytic DNA are rehydrated and they
can migrate on the membrane. If the flow buffer contains Pb2+, a cleavage reaction can occur on
the surface.

Adsorbant pad

C o lo r in test zo ne
(cap ture o f p ro d uct T est Z o ne
D ip test for P b(II) b y cap ture D N A) Membrane
C o ntro l Z o ne
Glass fiber 5' 3'

Pb (II) conjugate pad

Red color in control zone
3' 5' (uncleaved substrate
captured by streptavidin)
5' 3'
N o Pb (II) 17E enzym e

M o d ified sub strate

B io tin G o ld nano p article

R ed co lo r o nly in co ntro l
zo ne (uncleaved sub strate C ap ture D N A
cap tured b y strep tavid in)
Strep tavid in

Figure 14. Dipstick detection of toxic metal ions such as Pb2+ based on catalytic DNA and gold nanoparticles.

The 5' product of the substrate (containing the gold nanoparticles) will be free to move
forward and can be captured by a complementary DNA strand in the test zone, producing a red
band to indicate presence of Pb2+. Any uncleaved substrate will be captured beforehand by
streptavidin in the control zone. If there is no Pb2+ present, there will be no color in the test zone,
but the control zone will have a red color. Thus, the color in the control zone can show if the test
has been carried out properly. The main objective is to move all of the sophisticated sensing
chemistry behind the scene and minimize user operation to a simple step. Preliminary results
show this design can detect lead qualitatively in the lab (Figure 14).102

20
 viii. The Shelf-Life and Operation of the Sensor in a Real-World Setting. To apply the
sensors for on-site, real-time detection and quantification, the sensor stability has to be high. It is
well known that the stability of the DNA in solution is relatively low (no more than a few weeks).
However, we normally store the sensors in dried (e.g., lyophilized) form and it has been shown
in the PI’s lab and in other labs that the shelf lives of dried DNA sensors can be many months
and even years. The data in this report were from dried DNA received from an oligonucleotide
synthesis company. When we are ready to carry out our sensing application, aqueous solution is
added to dissolve the DNA, mix the sensor components, and conduct the sensing. The sensing
process completes in a few minutes, much shorter than the time required to degrade the DNA
sensors under normal conditions.

Figure 15. (A) Stability study of the catalytic DNA-based lead sensor after drying at different temperatures.
Stability at 45 oC (B) and 80 oC (C). (D) Stability of DNA attached to gold nanoparticles. The nanoparticles
were stored at room temperature in a glass vial on a work bench.

The dried sensor has high stability at room temperature. To shorten the time needed for
stability tests, we stored the sensor at extreme temperatures, such as -20 to 80 oC. The sensor did
not show much difference in terms of lead response in all the storage conditions (Figure 15A),
such as under -20ºC (6 days), room temperature (2 months), 45 ºC (21 days) and 60 ºC (6 days).
After storing at 45 oC for over 100 days, the sensor still maintained its activity (Figure 15B).
Even after storage at 80 oC, at which most antibody-based sensors will lose their functions, the
catalytic DNA-based sensor was still active (Figure 15C). This high stability allows the sensor to
be used in field applications, where extreme temperature conditions may be encountered. These
results demonstrate the excellent long-term stability of the dried sensors.

21
3. Summary and Outlook

In conclusion, we have demonstrated highly sensitive and selective fluorescent sensors for a
number of heavy metals, including Pb2+, UO22+, Cu2+, and Hg2+. Each sensor was highly
sensitive and selective towards its target metal ions. The use of the Pb2+ sensor to detect Pb2+ in
field water samples with a portable fluorometer has been demonstrated. Incorporation of
inorganic nanoparticles into DNAzyme nanostructures has proven to be a useful method to
design highly sensitive and selective colorimetric and fluorescent sensors. Because nucleic acids
can be selected for essentially any target molecule of choice, the methods of sensor design
described in this report should be applicable to the detection of many other analytes of interest.
Future work will be focused on increasing sensor sensitivity, selectivity, stability, and user-
friendliness. These improvements can be achieved by: performing detailed characterizations to
understand nucleic acid and nanoparticles interactions, introducing signal amplification
mechanisms, and introducing novel sensing platforms such as lateral flow devices. The
technologies demonstrated in this project have been licensed to DzymeTech, Inc., a local startup
company that will commercialize the sensor products in the near future.

22
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