Functional DNA Fluorescent and Colorimetric
Sensors for Heavy Metal ions and Endotoxins
Yi Lu
Department of Chemistry, Biochemistry, Bioengineering,
Materials Science and Engineering,
Center for Biophysics and Computational Biology, and
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
Urbana, IL 61801
A Grand Challenge Facing
Natural Resource Sustainability
On-site, real-time detection and quantification of toxic materials, such as
heavy metal ions and endotoxins, before, during and after remediation.
A Major Scientific and Technological Issue
Group 1
Period
2
1
1
H
2
Li Be
3
Na Mg
4
3
11
19
4
12
20
3
4
5
6
7
8
9
10
11
12
• Essential Metal ions
• Bulk and essential non-metal ions
• Non-essential biological elements
• Toxic metal ions
• Radionuclides
23
18
2
He
5
6
7
8
9
10
B
C
N
O
F
Ne
13
14
15
16
17
18
Al
Si
P
S
Cl
Ar
31
29
30
32
33
34
35
36
Zn Ga Ge
As
Se
Br
Kr
37
39 40
47
48
49
50
51
52
53
54
Ag Cd
In
Sn
Sb
Te
I
Xe
44
27
17
Cu
43
26
16
28
42
25
15
Sc Ti V Cr Mn Fe Co Ni
41
24
14
K Ca
38
21 22
13
45
46
5
Rb Sr
Y Zr Nb Mo Tc Ru Rh Pd
6
Cs Ba * Lu Hf Ta W Re Os Ir
55
71 72
7
Fr Ra ** Lr Rf Db Sg Bh Hs Mt Uun Uuu Uub Uut Uuq Uup Uuh Uus Uuo
87
56
88
73
74
75
76
77
78
79
80
81
82
83
84
85
86
Pt
Au Hg
Tl
Pb
Bi
Po
At
Rn
112 113 114
115
116
117 118
69
70
103 104 105 106 107 108 109 110
57 58
59
60
61
62
63
64
*Lanthanides * La Ce Pr Nd Pm Sm Eu Gd
89 90
91
92
93
94
95
96
111
65
66
Tb
Dy Ho Er
Tm Yb
98
99
101
Cf
Es Fm Md No
97
**Actinides ** Ac Th Pa U Np Pu Am Cm Bk
67
68
100
102
Toxic materials in the environment are large in numbers, subtle in structural
differences, small in quantities.
Therefore they represent new challenges and opportunities in natural
resource sustainability.
Instrumental Analyses
Mass spectrometry
Inductively Coupled Plasma
Advantages

Industrial standard
 Highly sensitive (down to
~ppb or less)
 Detect a number of
analytes simultaneously
Disadvantages
Require
sophisticated equipment, samplepretreatment, and skilled operators
difficult for in-situ, on-site, remote or
real-time detection/imaging
Four key steps in designing sensors
? a general method to obtain molecules for any specific
target (e.g., Lead, mercury, cocaine, endotoxins, cancer)
?
a general method to improve selectivity;
a general method to transform molecular
recognition into physical detectable signals without
compromising the binding affinity and selectivity;
?
?
a general method to fine-tune the dynamic range.
Four key steps in designing sensors
? a general method to obtain molecules for any specific
target (e.g., Pb2+, Amphetamine, cocaine, Ricin, cancer)
virus
Metal ions
pH
Method
toxins
bacteria
Organic cofactors
Until recently, antibody is the only method that is general enough for a broad
range of targets, but antibodies are not very good at sensing small molecules.
Functional DNA
RNA
DNA
O
5'
A
O
O
DNA/RNA = Protein enzymes
DNA/RNA = Antibodies
A
O
2'
3'
H
O
O P O
OH
O

O P O
O
C
O
Substrate

C
O
M(II)
A: DNAzyme
O
Enzyme
H
O
OH
O
O P O
+
O P O
G
O
G
O
O
O
H
O
O P O
OH
O

O P O
T
O
B: Aptamer
U
O
O
O
H
O
O P O
O

C: Aptazyme
OH
O

O P O
O

M(II)
H
H
N
N H
N
O
N
N
N
H N
N
N H
O
T
N
N
N
N
A
H N
O
N H
O
G
C
H
Kruger, K. et al. Cell 31, 147 (1982).
Guerrier-Takada, C. et al. Cell 35, 849 (1983).
Breaker, R.; Joyce, G. Chem. Biol. 1, 223 (1994).
Combinatorial biology: a general method to
obtain DNA/RNA for a specific target
In vitro Selection
Systematic Evolution of Ligands by Exponential Enrichment (SELEX)
Advantages:
• High throughput (1014-1015
different sequences)
• Selective Amplification
• Improvement in each round
• Minimal cost
• Short time
Ellington, A. D.; Szostak, J. W. Nature 346, 818(1990).
Tuerk, C.; Gold, L. Science 249, 505 (1990).
Beaudry, A. A.; Joyce, G. F. Science 257, 635 (1992).
Molecules Recognized/bound by Selected DNA/RNA
Analyte/target type
Examples
Metal ions
Mg(II), Ca(II), Mn(II), Pb(II), Hg(II), U(VI)
Organics
Cibacron blue, reactive green 19
Amino acids
L-Valine, D-Tryptophan
Nucleosides/nucleotides
Guanosine, ATP
Nucleotide analogs
8-oxo-dG, 7-Me-guanosine
Biological cofactors
NAD, FMN, porphyrins, Vitamin B12
Aminoglycosides
Tobramycin, Neomycin
Antibiotics
Streptomycin, Viomycin
Peptides
Rev peptide
Enzymes
Human Thrombin, HIV Rev Transcriptase
Growth cofactors
Karatinocyte GF, Basic fibroblast GF
Antibodies
human IgE
Gene regulatory factors
elongation factor Tu
Cell adhesion molecules
human CD4, selectin
Intact viral particles
Rous sarcoma virus, Anthrax spores
Juewen Liu and Yi Lu, J. Fluoresc. 14, 343-354 (2004).
Examples of in vitro selected DNAzymes
3' N N N N N N Y rR N N N N N N N 5'
5' N N N N N N R
N N N N N N N 3'
A
G
G
G
C
C
T
A
A
A
G
C
CTA
3' N N N N N N G rN N N N N N N N 5'
5' N N N N N N T
N N N N N N N 3'
AA
C
C
G
G
G
A
G TC
G C
C
Mg2+
Pb2+
UO22+
Zn2+
5' Y Y Y Y A A T A C G N N N N N 3'
R R R R A C 3'
YYYY
C
CGG
C N N N N N 5'
T
G
Cu2+
Lu, Y. Chem. Euro. J. 8, 4588-4596 (2002).
Co2+
Examples of in vitro selected DNAzymes
3' N N N N N N Y rR N N N N N N N 5'
5' N N N N N N R
N N N N N N N 3'
A
G
G
G
C
C
T
A
A
A
G
C
CTA
3' N N N N N N G rN N N N N N N N 5'
5' N N N N N N T
N N N N N N N 3'
AA
C
C
G
G
G
A
G TC
G C
C
UO22+
Mg2+
Pb2+
Substrate
M(II)
Enzyme
5' Y Y Y Y A A T A C G N N N N N 3'
R R R R A C 3'
YYYY
C
CGG
C N N N N N 5'
T
G
Cu2+
General secondary structure, making it easy for sensor design
Lu, Y. Chem. Euro. J. 8, 4588-4596 (2002).
Why using functional DNA in sensing?
 environmentally benign
 cost effective
 stable under rather harsh conditions
 DNA is ~1,000-fold more stable to
hydrolysis than proteins and ~ 100,000fold more stable than RNA
 Globular shape:
• More resistant to nucleases
• Less likely to bind other molecules
 can be denatured and renatured many times
(for manufacturing and storage)
 Non-immunogenic
 Can be readily modified for signal transduction
 can be genetically engineered to be delivered
to a specific location
Antibody
Functional DNA
Juewen Liu, Zehui Cao, and Yi Lu, Chem. Rev. 109, 1948-1998 (2009).
Four key steps in designing sensors
√ a general method to obtain molecules for a specific
analyte;
?
a general method to improve selectivity;
What most want to do
What most end up doing
Problems with Selectivity in in vitro Selection
0.12
0.1
kobs (min-1)
0.5 mM Co(II)
0.08
0.5 mM Zn(II)
0.5 mM Pb(II)
0.06
0.04
0.02
0
No Negative Selection
•
•
Sequences selected with Co2+ show high activity with Zn(II) and Pb(II)
Must reduce or remove unwanted activity to improve metal selectivity
Initial pool of
DNA/RNA
2. A general method to
improve selectivity:
negative selection
Selections
using only Co
2+
DNA/RNA with
2+
Co activity
Positive
Selection


Incorporate negative
selection to reduce
“unwanted” activity
Can be performed in
parallel with positive
selection for
comparison
Selection
using only Co
2+
Negative
Selection
Selection with a "metal
soup" containing
competing metal ions
Remove sequences that are
active with other metal ions
2+
High Co activity,
2+
Continue Co selection
2+
but with low Co
P. J. Bruesehoff, J. Li, A. J.
Augustine III, and Y. Lu,
Combinatorial Chemistry and
High Throughput Screening,
5, 327-335 (2002).
selectivity
2+
High Co activity with
2+
high Co selectivity
Improved Metal Ion Selectivity after
Negative Selection
0.25
0.2
0.5 mM Co(II)
kobs (min-1)
0.5 mM Zn(II)
0.15
0.5 mM Pb(II)
0.1
0.05
0
No Negative Selection
Negative Selection
P. J. Bruesehoff, J. Li, A. J. Augustine III, and Y. Lu, Combinatorial Chemistry
and High Throughput Screening, 5, 327-335 (2002).
Four key steps in designing sensors
√ a general method to obtain molecules for a specific
analyte;
√ a general method to improve selectivity;
? a general method to transform molecular
recognition into physical detectable signals without
compromising the binding affinity and selectivity;
Analytes
Signal
Target
Signal
recognition
transduction
part
part
DNAzyme/
Aptamers
Fluorescence
Color
Turn analyte-dependent catalytic reactions
into physically detectable signals
A general method to convert DNAzymes into fluorescent
sensors using catalytic beacon
cleavage site
substrate strand 17DS
3'- G T A G A G A A G G rN T A T C A C T C A -5'
5'- C A T C T C T T C T
A T A G T G A G T -3'
Rh-17DS
A A
C
C
G
G
T CG
A
G
enzyme strand 17E
C
G C
17E-Dy
COO3' 5'
17DS
5'
17E
O
O
3'
O
P
TAMRA
O
O
O
N N
N
H
OH
N
Dabcyl
Fluorescent
tag
Hybridization
with catalytical DNA
Fluorescence
suppressed
rA
Catalytic DNA
strand
Quencher
Pb2+
rA
80000
fluorescence intensity
rA
Catalytic DNA
strand
H
N
O
O P O
O-
O-
RNA linkage
Substrate strand
N+
O
N
I
III
+Enz
60000
II
I
+Pb2+
40000
III
20000
II
0
570
600
630
660
Quencher
wavelength (nm)
Li, J.: Lu, Y. J. Am. Chem. Soc., 122, 10466-10467. (2000).
690
A general method to convert DNAzymes into fluorescent sensors using
catalytic beacon for a broad range of metal ions
Fluorescent sensors
Based on catalytic beacon
Pb2+: Detection limit: 1 nM
EPA MCL:
75 nM
UO22+: Detection limit: 45 pM
EPA MCL:
126 nM
ICP-MS:
420 pM
Cu2+: Detection limit: 35 nM
EPA MCL:
20 μM
Hg2+: Detection limit: 2.4 nM
EPA MCL:
10 nM
Li, J.: Lu, Y. J. Am. Chem. Soc., 122, 10466-10467. (2000).
J. Liu, et al. Proc. Natl. Acad. Sci 104, 2056 (2007).
J. Liu and Y. Lu J. Am. Chem. Soc. 129, 9838 (2007).
J. Liu and Y. Lu, Angew. Chem., Int. Ed., 46,7587 (2007).
High specificity or selectivity
Over 1 million fold selectivity over Th(IV),
and hundreds of millions fold selectivity over other metal ions
Juewen Liu, Andrea K. Brown, Xiangli Meng, Donald M. Cropek, Jonathan D. Istok, David B. Watson, and Yi Lu,
Proc. Natl. Acd. Sci. USA 104, 2056 (2007).
Design of a Simple Colorimetric Biosensor
cleavage site
substrate strand 17DS
3'- G T A G A G A A G G rAPb
T A T C A C T C A -5'
5'- C A T C T C T T C T
A T A G T G A G T -3'
A A
C
C
G
G
T CG
A
G
enzyme strand17E
C
G C
BLUE
Mix
Aggregate
Pb
RED
Pb
17E
=
5'GGAAGAGATG
Au
=
Pb
BLUE
=
S
rA
-3' =
SubAu
5'-CACGAGTTGACA-3'
Pb
RED
= DNAAu
Liu, J.; Lu, Y. J. Am. Chem. Soc. 125, 6642-6643 (2003).
pH Indicator like Operation
0
0.3
0.5
5 M Mg(II) Ca(II)
pH 9.5
Mn(II)
8.0
1
Co(II)
7.0
2
3
4
Ni(II)
Cu(II)
Zn(II)
4.0
3.0
2.0
Liu, J.; Lu, Y. J. Am. Chem. Soc. 125, 6642-6643 (2003).
Liu J.; Lu, Y. Chem. Mater., 16, 3231 (2004);
Liu J.; Lu, Y. J. Am. Chem. Soc. 126, 12298 (2004).
5 M Pb(II)
Cd(II)
1.0
Colorimetric sensors for Uranyl
Detection limit: 50 nM
EPA MCL:
130 nM
1 nM
Lee, J.-H.; Wang, Z.; Lu, Y. J. Am. Chem. Soc. 130, 14217-14226 (2008).
Wang, Z.; Lee, J.-H.; Lu, Y. Adv. Mater., 20, 3263–3267 (2008).
Four key steps in designing sensors
√ a general method to obtain molecules for a specific
analyte;
√
a general method to improve selectivity;

a general method to transform molecular
recognition into physical detectable signals without
compromising the binding affinity and selectivity;
?
a general method to fine-tune the dynamic range.
The need for tunable dynamic range
• 22 million old houses in the US alone have used lead paint
• US federal “thresholds” are 1.0 mg/cm2 and 0.5%
• Current lead detection kits are based on Na2S9H2O or
sodium rhodizonate.
• A study of available kits showed low rates of both false positive
and false negative results when compared to laboratory
analytical results using the federal thresholds (www.hud.gov)
• Current kits cannot tune the dynamic range
Threshold
yes
Signal
yes
no
no
Lead concentration
IDEAL CURVE
Threshold
A Colorimetric Biosensor
with Tunable Dynamic Range
A. Active DNA
cleavage site
substrate strand 17DS
3'- G T A G A G A A G G rA T A T C A C T C A -5'
Extinction
Extinction
1.5
1.5
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 C
A T A G T G A G T -3'
A A
C
C
G
G
T CG
A
G
enzyme strand17E
C
G C
0.5
0.5
0.0
0.0
15
15
C
C
12
12
EE522
/E
/E700
522
700
cleavage site
substrate strand 17DS
B
B
450
450
550
650
750
450 550
550 650
650 750
750
450
550
650
750
Wavelength
Wavelength
Wavelength (nm)
(nm)
Wavelength (nm)
(nm)
5'- C A T C T C T T C T
A T A G T G A G T -3'
A A
C
C
G
G
T CG
A
G
enzyme strand17E
C
G C
B. Inactive DNA
1.0
1.0
A
A
9
9
B:A = 20:1
B:A = 0:1
6
6
3
3
0
0
0.01
0.01
0.1
0.1
1
1
[Pb(II)]
[Pb(II)] M
M
Brown, A. K.; Li, J.; Pavot, C. M.-B.; Lu, Y. Biochemistry 42, 7152-7161 (2003).
Liu, J.; Lu, Y. J. Am. Chem. Soc. 125, 6642-6643 (2003).
10
10
100
100
Pb2+ Detection in Paint
1.0 mg/cm2 (threshold)
qualitative
0
0.05
0.34
1.22
0.81
4.71
1.52
mg Pb / cm2 paint
10
8
Latex paint
Oil paint
7
Latex on latex
Oil on oil
Oil on Latex
Latex on Oil
8
A538/A700
A538/A700
6
5
4
3
6
4
2
2
1
Portable colorimeter
quantitative
0
0
0
1
2
mg/cm2 Pb
3
4
5
0
1
2
3
4
2
mg/cm Pb
5
6
7
The tunable sensor allowed our Pb sensor to change color
right at the federal threshold of 1.0 mg/cm2, and to work with
different types of paints.
Beyond metal and catalytic DNA-based detection
Metal ions (Mg(II), Ca(II), Pb(II), Zn(II))
Organic dyes (Cibacron blue, reactive green 19)
Amino acids (L-Valine, D-Tryptophan)
Nucleosides/nucleotides (Guanosine, ATP)
Nucleotide analogs (8-oxo-dG, 7-Me-guanosine)
Biological cofactors (NAD, FMN, porphyrins, Vitamin B12)
Aminoglycosides (Tobramycin, Neomycin)
Antibiotics (Streptomycin, Viomycin)
Peptides (Rev peptide)
Enzymes (Human Thrombin, HIV Rev Transcriptase)
Growth cofactors (Karatinocyte GF, Basic fibroblast GF)
Antibodies (human IgE)
Gene regulatory factors (elongation factor Tu)
Cell adhesion molecules (human CD4, selectin)
Intact viral particles (Rous sarcoma virus, Anthrax spores)
Juewen Liu and Yi Lu, J. Fluoresc. 14, 343-354 (2004).
Endotoxins
• Endotoxins - potentially toxic natural compounds found in the outer cell
membrane of various gram-negative bacteria
• Also known as LPS (LipoPolySaccharides) and consist of a polysaccharide
chain and a lipid moiety
• Humans exposed to endotoxins either through ingestion and inhalation of
gram negative bacteria either in medical environments or in industrial
settings
• Ailments including skin rashes, malaise, fevers and respiratory distress
attributed to endotoxin exposure
• USP and FDA standard is the Limulus Amebocyte Lysate (LAL) gel-clot
method, which suffers from interferences due to large number of
contaminants present in metalworking fluids, common in industrial
environments
Limulus polyphemus:
commonly known as
Horseshoe-crab
Structure of Endotoxins (or LPS)
a)
Inner
Core
Outer
Core
O-Specific Chain
General architecture :
Lipid A
Lipid
Polysaccharide
CO2-
b)
Structural representation :
Kdo
CO2GluN
Dgalactose
heptose
PO4DGluN
Kdo
CO2-
Dglucose
DDgalactose glucose
heptose
PO4-
Polysaccharides
KDO : 2-keto-3-deoxyoctulosonic acid
Present in LPS expressed by most
gram-negative bacteria
heptose
PO4-
Kdo
DGluN
PO4Lipid
Selection progress
2.5
Switching Activity
2
Endotoxin
KDO
1.5
1
0.5
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Selection Round
• Switching Activity = Ratio of radiation of eluted fractions over background
• Round 1-15 : small increase over background ; R16 : double the background
Jennifer Deluhery and Nandini Nagraj
A general method to turn aptamers into
colorimetric sensors
Liu J.; Lu, Y. Angew. Chem., Int. Ed., 45, 90-94 (2006).
Liu, J.; Lu, Y. Nature Protocol 1, 246-252 (2006).
A dipstick test using functional DNA
nanoparticles
Cocaine
Juewen Liu, Debapriya Mazumdar and Yi Lu, Angew. Chem., Int. Ed. 45, 7955 –7959 (2006).
Summary
 To obtain sensors for a broad range of targets, general
strategies have been developed to
•
•
•
•
to obtain sensing molecules;
to improve selectivity;
to convert molecular recognition event into physically detectable signals;
to tune the dynamic range.
 The combination of functional DNA and nanotechnology
resulted in sensors that
• are stable and cost-effective.
• are highly sensitive and selective.
• have tunable dynamic range
• can be applied to detection and quantification of a broad range of
targets (metal ions, organic molecules, proteins and cells….)
• allow real-time, on-site (or remote) sensing.
Acknowledgments
DNA/RNA Lab at UIUC
Collaborators
Dr. Jennifer Deluhery, Dr. Kishore Rajagopalan (ISTC)
Graduate Students:
Margaret Shyr, Dr. Paul Braun (MatSE, UIUC)
Ying He
Andrea K. Brown
Abhijit Mishra, Gerard C. L. Wong (MatSE, UIUC)
Hannah Ihms
Peter J. Bruesehoff
Rong Tong and Jianjun Cheng (MatSE, UIUC)
Tian Lan
Hee-Kyung Kim
Tae-Jin Yim, Dr. Jonathan S. Dordick (CheE, RPI)
Darius Brown
Jing Li
Carla B. Swearingen, Dr. Paul W. Bohn (Chem, UIUC)
Nandini Nagraj
Jung Heon Lee
Kashan A. Shaikh, Dr. Chang Liu (ECE, UIUC)
Eric Null
Juewen Liu
In-Hyoung Chang, Dr. Donald M. Cropek (CERL, DoD)
Zidong Wang
Kevin E. Nelson
Brian Wong
Daryl P. Wernette
Funding:
Weichen Xu
Mehmet V. Yigit
ISTC, EPA, HUD, NIH, DOE, NSF, & DOD
Hang Xing
Debapriya Mazumdar
Yu Xiang
Seyed-Fakhreddin Torabi
Postdoctoral fellows:
Hui Wei
Zehui Cao
Juanzuo Zhou
Geng Lu
Xiangli Meng
Daisuke Miyoshi
Wenchao Zheng
Selection Strategy
biotin
5'
avidin-coated bead
5'
5'
P1
P1
P2
annealing 1
5'
5'
5'
5'
5'
annealing
5'
2
5
R
1. NaOH
2. PAGE
P2
5'
1. Incubation
2. Elution
5'
5'
5'
5'
4
3
5'
PCR
5'
R
5'
• Selection with 1.25 µg/mL endotoxin in 50 mM HEPES-Na at pH 7.35 ,
containing 50 mM NaCl, 5 mM KCl and 5 mM MgCl2
• Parallel positive selection with KDO to expand selectivity and
sensitivity of aptamers to most gram-negative bacterial strains
In vitro selection
In vitro selection with [Mn+]
as metal ion cofactor
N40
PCR
B
rA
N40
+
rA
B
B
Metal cofactor
B
Cloning and sequencing
PCR
Introducing mutations
rA
rA
Further selection by decreasing
reaction time and [Mn+]
NaOH
B
rA
Cloning and sequencing
R. R. Breaker, G. F. Joyce Chem. & Biol. 1, 223 (1995).
J. Li, W. Zheng, A. H. Kwon, and Y. Lu
Nucleic Acids Res. 28, 481 (2000).
Characterization
This method is adaptable to any metal ion and any oxidation state of the same metal ion
with tunable activity, and affinity
In vitro selection
Liu, J. et al. Proc. Natl. Acad. Sci 104, 2056 (2007).
Future Goals
• Continuation and comparison of selection
with both endotoxin as well as KDO
• Incorporation of negative selection with
metalworking fluids to increase selectivity
• Variation of incubation time to further
increase sensitivity
Selection Progress : Gel-based assays
LPSR10
M
KDOR10
PAGE gels after every round
of PCR amplification to
separate bound sequences
from unbound sequences