DNA-based biosensors for metal detection

Juewen Liu

Department of Chemistry Waterloo Institute for Nanotechnology

University of Waterloo

June 6, 2018

GWF meeting (Hamilton)

Problem to solve: heavy metal contamination

Importance and challenges of metal detection

http://userpage.chemie.fu-berlin.de

• Environmental monitoring

• Food analysis

• Biomedical diagnosis

http://www.nanoscience.imdea.org

• Instrumentation analysis1. Highly accurate, reliable2. Multiplexed detection3. Industrial standard4. Expensive5. Long turnaround time6. Detects total metal

• (Bio)sensors1. Cost effective2. On-site, real-time detection3. Detects bioavailable metal

Various metal ligands

Hg2+ Zn2+

Nat. Chem. Biol. 2008, 4, 168-175

Small molecule fluorescent chelators Peptides, proteins, & antibodies

Genetically engineered cells Nucleic acids

Coord Chem Rev, 256, 2012, 2175 Curr Opin Chem Biol, 12, 2008, 214

http://2010.igem.org/Team:Peking/Project/Biosensor

UO22+

Highly stable, cost effective, easy to modify, combinatorial selection + rational design

DNA for metal binding

Sigel, R. K. O.; Sigel, H. Acc. Chem. Res. 2010, 43, 974.

• Phosphate: electrostatic interaction, binds hard Lewis acids

• Bases: metal coordination

In vitro selection of a cerium-dependent DNAzyme

rAF Ce4+

PCR1

1

2 PAGEN50

P2

P1

PCR2rAFP3

P4

PAGE 345

119 nt 91 nt

P. J. Huang, J. Lin, J. Cao, M. Vazin and J. Liu*, Analytical Chemistry, 86, 1816-1821 (2014)

1013-1014 DNA sequences

Ce(NH4)2(NO3)6

Selection progress

Round 1 Round 2 Round 3

9.3%0.6%

82.1%

99.0%

99.9%

Round 4

10.7%

Round 5

25.2%

74.0%

Round 6

26.8%

71.4%87.8%

Sequence alignment

AAATGATCCACTAATACGAGTCACTATrAGGAAGATG

TTCTACCAGTGAT

GCGA

AA

N50

5’

Cleavage site Start of the N50 region

-ACGAGTCACTATrAGGAAGATGGC

TACCG-TG CAGTGATA

GAAAC

AGGTT

5’

A CCT

TTGAA GT

CAGC

GGTCG

C

TA

TA

TACT

AGGTCAA

GTGGGTGG A

Ce13

GTCACGAGTCACTATrAGGAAGATGGCGAAA

TACCGCTTTCAGTGCTCAGTGATATT AGGTCA

AGTGGGTGG A

Ce13d

5’

A general lanthanide-dependent DNAzyme

• Y3+ shows similar activity like other lanthanides• Pb2+ is the only active divalent metal ion

Left lane: 10uM Right lane: 100uM

La3+ Ce3+ Ce4+ Pr3+ Nd3+ Sm3+ Eu3+ Gd3+ Tb3+ Dy3+ Ho3+ Er3+ Tm3+ Yb3+ Lu3+

Sr3+ Fe3+ Y3+ In3+ Tl3+

Mg2+ Mn2+ Fe2+ Co2+ Cu2+ Zn2+ Hg2+ Pb2+

P. J. Huang, J. Lin, J. Cao, M. Vazin and J. Liu*, Analytical Chemistry, 86, 1816-1821 (2014)

Highly sensitive lanthanide detection

t (min)

0 20 40 60 80 100 120

F (a

.u.)

120

150

180

210

240

270

300

-MCH+MCH

t (min)

0 20 40 60 80

F (a

.u.)

100

130

160

190

220

250

280

0

5 nM10 nM

20 nM50 nM0.1 µM0.5 µM1 µM

[Ce3+] (nM)

0 200 400 600 800 1000

dF/d

t

0

3

6

9

12

15

0 10 20 30 40 50012345

t (min)

0 10 20 30 40 50

F (a

.u.)

90

120

150

180

210

240

270

300

No metalCe3+

Sc3+

Fe3+

In3+

Tl3+

t (min)

0 10 20 30 40 50

F (a

.u.)

90

120

150

180

210

240

270

300

No metalCe4+

Y3+

La3+

Ce3+

Pr3+

Nd3+

Sm3+

Eu3+

Gd3+

Tb3+

Dy3+

Ho3+

Er3+

Tm3+

Yb3+

Lu3+

F Ce3+

Q Q

Ce3+Pb2+

OH

SH

MCH

P. J. Huang, J. Lin, J. Cao, M. Vazin and J. Liu*, Analytical Chemistry, 86, 1816-1821 (2014)

Characterization by phosphorothioate (PS) modifications

rAGGAAG

TT AGGTCA

AGTGGGTGG A

OO

O

O-

P

O

O OH

A

G OO

O

S-

P

O

O OH

A

G

Ce Mg Mn Fe Co Ni Cu Zn Cd Hg PbCe13d

PO

PS

So, it’s just another DNAzyme？

Anything special about this one?

Ce13d with PS substrate can detect thiophilic metals as a group.P Huang and J Liu, Analytical Chemistry, 86, 5999–6005 (2014).

Selection of DNAzymes using a PS-modified library

OO

O

S-

P

O

O OH

A

GFAM-AAATGATCCACTAATACGAGTCACTATrAGGAAGATG

TTCTACCAGTGAT

GCGA

AA

N50

5’

Can we get new DNAzymes that can discriminate different thiophilic metals?

Single modification made at the fixed region.

Perrin, Angew. Chem. Int. Ed. 2008, 47, 4346 –4350

So far, DNAzymes for soft metals are rare.

Solution 1: modified bases Solution 2: modified phosphate backbone

A highly Cd2+-specific DNAzyme

Cd2+ Pb2+ Cu2+ Ce3+

Rat

e (m

in-1

)

0.00

.02

.04

.06

.08

.10

.12

.14

Red bars = Ce13d

GTCACGAGTCACTATrA*GGAAGATGGCGAAA

T-CCTTCTACCGCTTTCAGTGCTCAGTGATA

5’

A TA CA GT ATGAT

5’BN-Cd16

Substrate

Enzyme

Black bars = new enzymeGTCACGAGTCACTATrAGGAAGATGGCGAAA

TACCGCTTTCAGTGCTCAGTGATATT AGGTCA

AGTGGGTGG A

P Huang and J Liu, Nucleic Acids Research, 2015

Highly sensitive Cd2+ detection

RefMg2+

Ca2+Mn2+

Fe2+Co2+

Ni2+Cu2+

Zn2+Ba2+

Hg2+Pb2+

Cd2+

dF/d

t (a.

u.)

0.0

.2

.4

.6

.8

1.0

1.2

1.4

100 nM1 M

t (min)

0 10 20 30

F (a

.u.)

15

20

25

30

35

40t (min)

0 10 20 30 40

F (a

.u.)

10

15

20

25

30

35

40

0 nM 2 nM5 nM10 nM20 nM50 nM100 nM200 nM

A

C

[Cd2+] (nM)

0 100 200 300 400 500

dF/d

t (a.

u.)

0.0

.5

1.0

1.5

2.0

2.5

3.0

0 10 20 30 40 500.0

.3

.6

.9

1.2

B

D

Cd2+

Other metals

FCd2+

F

Q

P Huang and J Liu, Nucleic Acids Research, 2015

Highly sensitive Cu2+ detection

OO

O

S-

P

O

O OH

A

G

SubstrateFAM-AGTCACTATrA*GGAAGATGGCGAAC

CTTCTACCGCTTGQ-CAGTGATA

5’

A AA CG CGAPSCu10

DNAzyme

Cu2+

Dete

LOD = 1.6 nM Cu2+

Huang P and Liu J, Anal. Chem. 88, 3341–3347, 2016

Highly sensitive Ag+ detection (using normal DNA)

GTCACGAGTCACTATrAGGAAGATGGCGAAA

TTCTACCGCTTTCAGTGCTCAGTGATAT

5’

G C G A C TG GG AG

GAG

AC

T

A TT

T

AA

A

GCGAC

G

C

TC

TT AG

Pb2+

No Metal Ag

+ K+ Li+ Tl+

Mg2+

Ca2+

Ni2+

Cu2+

Zn2+

Co2+

Sr2+

Cd2+

Mn2+

Hg2+

Fe2+

Fe3+

Ce3+

Ga3+

Tl3+

Y3+

Cle

avag

e (%

)

0

10

20

30

40

50

60

7010 M Mn+

100 M Mn+

Cle

avag

e(%

)

0

20

40

60

80NaNO3 NaCl

[Na+] (mM)

0

12.5 25 50 100 200 300 400 500

750 1000

Saran R. and Liu J. Anal. Chem. 2016, 88, 4014-4020

Monovalent, thiophilic Ag+, but it works very well!This is a surprise!

Only 52 sequences out of >50,000

Efficient, 0.41 min-1 Time (min)0 20 40 60

Rel

ativ

e Fl

uore

scen

ce

1.0

1.5

2.0

2.5

3.0 500 nM 400 nM 300 nM200 nM160 nM

25 nM

120 nM 100 nM 50 nM

0 nM

Detection limit = 24 nM Ag+

EtNa has the best Ca2+ specificityA

C D

EtNa

E5 10-23

B

8-17 variants

ChemBioChem, 18, 518-522. (2017)

EtNa binds two Ca2+ but only one Mg2+

Slope = 2

Slope = 1

ChemBioChem, 18, 518-522. (2017)

C

Ca2+ detection

Limit of Detection = 17 µM Ca2+

A DNA code for metal ions

• Many new metal-specific DNAzymes isolated.

• Please start to use them.

Wenhu Zhou, Runjhun Saran and Juewen Liu, Chemical Reviews, 117, 8272–8325 (2017)

Acknowledgments

$$: University of Waterloo, NSERC, CIHR, CFI, Ontario MRI, CFREF

•Postdoctoral FellowDr. Xu ZhangDr. Feng Wang

•Graduate StudentsNeeshma DavePo-Jung Jimmy HuangAjfan BaessieAlexander IPImran KhimjiBiwu LiuHoward TsaiZiyi SunMahsa VazinRunjhun SaranWenhu ZhouLingzi MaZhicheng HuangUndergraduates

Rachel Pautler Ahmed Zaki David Lin Jeehae ShinThomas Kennedy Anand Lopez Erin Kelly Firas El-HamedJames MacLean Nathan Lang Marissa WuNish Bhatt Kevin Joseph Murat KiyBrendan Smith Kiyoshi Morishita Youssef HelwaArsalan Beg Menhaj Jenny Lin Zachary Jacobi

Visiting Students/ScholarsSylwia WalczkZenata MatuszekElsa MateronDr. Hui Jiang

Cr3+, Na+, Ca2+

Fe3+, Ln3+, Cu2+, Cd2+

Ag+