Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801

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Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Yi Lu
        Department of Chemistry
University of Illinois at Urbana-Champaign
             Urbana, IL 61801
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
A major challenge facing ERSP/SBR
   Measurement/monitoring of radionuclides and metal ions
          across a large spatial and temporal scale

                  cellular level                 ecological level
Before ER: How many and how much (identification and quantification)?
        Where and when (on-site, real-time with high spatial and temporal resolution)?
        What species (e.g., different oxidation states with different bioavailability)?
During ER: How effective are the remediation methods?
After ER: Long-term monitoring (legacy management)
Fundamental science: Mechanistic studies (fundametnal process coupling)
         Computer modeling (the more accurate data, the better simulation)              More
selective chelators (structural features responsible for binding
        different radionuclides and different oxidation states of the same radionuclide)
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Current instrumental analysis
      Atomic absorption spectrometry
      Inductively coupled plasma mass spectrometry
      Anodic stripping voltammetry
      X-ray fluorescence spectrometry or microscope
      Phosphorimetry
  Advantages              Disadvantages
   Industrial standard         Require expensive equipment, sample-
  Highly sensitive (down to
                                          pretreatment, and skilled operators
           ~ppb or less)       Detect total amount of metal ions

  Detect a number of metal                (not bioavailable metal ions)
 ions simultaneously            cannot differentiate different oxidation states
                                          of the same metal ions (U(VI)/U(IV);
                                          Cr(VI)/Cr(III); Fe(III)/Fe(II))
                                difficult for in-situ, on-site, remote or
                                          real-time detection
Designing highly sensitive and selective sensors is a solution
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Number of publications in metal ion sensing

                   ~50,000 publications as of 2009

There are many publications and thus research activities in metal
sensing….but very limited practical or commercially available sensors.
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Why are there so few metal ion sensors that
                are being used practically?

The process is too much of a trial-and- error process and lack
    ? a general method to obtain molecules for any specific
    metal ions and any specific oxidation state of metal ions
    ?   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.

                What can we do about it?

We need to design general strategies to meet the four key
challenges in metal ion sensing.
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Four key steps in designing sensors
     ? a general method to obtain molecules for any
     specific target (e.g., U(VI), Cr(VI), Cr(III), organic
     contaminants, proteins, bacteria, viruses)
                                   U(VI)
                                                       Cr(III)
           Cr(VI)

                                 Method

                                                         proteins
             bacteria
                             Organic contaminants

Until recently, antibodies (Abs) is the only method that is general enough for a
broad range of targets, but Abs not very good at sensing small molecular
targets or targets that are either non-immunogenic or too toxic to raise Abs.
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Functional DNA: catalytic DNA and aptamers

                   DNA/RNA = Protein enzymes
                   DNA/RNA = Antibodies

                      Catalytic DNA

                        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).
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Combinatorial biology: a general method to
   obtain DNA/RNA for a specific target
In vitro Selection
Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

                           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).
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
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).
Yi Lu Department of Chemistry University of Illinois at Urbana-Champaign Urbana, IL 61801
Examples of in vitro selected Catalytic DNA

                                                                            UO22+
                          Mg2+                                               (Lu)
                         (Joyce)

                          Pb2+                                             Zn2+
                          (Lu, Joyce)                                      (Lu)

                           Cu2+                                             Co2+
                         (Breaker)                                          (Lu)

Specific sequence/code
= specificity
                                        Lu, Y. Chem. Euro. J. 8, 4588-4596 (2002).
Examples of in vitro selected catalytic DNA

                                                                   UO22+
                     Mg2+

                       Pb2+
                                 Substrate
                                                           M(II)

                                Enzyme

                     Cu2+

General secondary structure, making it easy for sensor design
              Lu, Y. Chem. Euro. J. 8, 4588-4596 (2002).
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
2. A general method to improve sensor selectivity

                             Applying a “negative” selection
                             strategy in in vitro selection or
                             SELEX, one can improve sensor’s
                             analyte selectivity. This method can
                             be generally applied to any
                             selection method.

                          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;
3. A general method to convert catalytic DNA into
fluorescent sensors using catalytic molecular beacon

                                                                             TAMRA

                                                                             Dabcyl

                                                                       I
                                                         III                  +Enz
                                                                       II
                                                          I
                                                                              +Pb2+

                                                                       III

                                                         II

         Li, J.: Lu, Y. J. Am. Chem. Soc., 122, 10466-10467. (2000).
A highly sensitive and selective sensor for Uranium

                 Over 14 fold fluorescence increase,
          with a Detection limit: 45 pM = 11 part-per-trillion
                   Over 1 Million Fold Selectivity
 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, 2056–2061 (2007).
Uranyl Detection in Soil Samples at Oak Ridge IFRC

               300×diluted                                                 50×diluted
     Catalytic DNA sensor response                                Phosphorescence in 10% H3PO4

 We followed a sample extraction procedure established by P. Zhou and B. Gu,
 Environ. Sci. Technol. 39, 4435-4440 (2005).
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, 2056–2061 (2007).
Further comparison

                                                            Method              Detection      Detection
                                                                                Limit (pM)     Limit (ppt)
                                                      X-ray Fluorescence        11,760,000       2,800,000
                                                       Atomic Absorption           336,000          80,000
                                                         Spectrometry
                                                            EPA MCL                126,000          30,000
                                                     ICP-Atomic Emission              8,400          2,000
                                                        Spectrometry
                                                     Antibody fluorescence            1,000            238

                                                          ICP-Mass                      420            100
                                                         Spectrometry
                                                     Stripping Voltammetry              100              24
                                                     Catalytic DNA Sensor                45              11
Performance is comparable to
                                                        Phosphorimetry                   42              10
 ICP and phosphorescence
                                                           Kinetic                       4.2              1
                                                        Phosphorimetry

 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, 2056–2061 (2007).
The method is general:
more catalytic beacon based fluorescent sensors

                                                          Pb2+: Detection limit: 1nM
  Fluorescent sensors                                           EPA MCL:      75 nM
Based on catalytic beacon

                                                          UO22+: Detection limit: 45 pM
                                                                EPA MCL:        126 nM

                                                          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).
Design of a Simple Colorimetric Biosensor

                   Pb
                                                                   BLUE

      Mix
                                 Aggregate
                                                                                Pb
RED
                                                                          Pb         Pb
      =     17E                                    BLUE                        Pb

            5'-             rA
      =        GGAAGAGATG             -3'    =   SubAu
                                                                   RED
              Au            5'-CACGAGTTGACA-3'           = DNAAu
      =
                                    Liu, J.; Lu, Y. J. Am. Chem. Soc. 125, 6642-6643 (2003).
Colorimetric Uranium Sensors

                                           Detection limit: 50 nM

                                            Detection limit: 1 nM
      Lee, J.H; Wang, Z; Liu, J; Lu, Y. J. Am. Chem. Soc. 130, 14217-14226 (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.

                     Threshold #2: water       Threshold #1: soil

                    yes                  yes
      Signal

               no          no

                    Lead concentration
                    IDEAL CURVE
4. A general method to tunable Dynamic Range

 A. Active DNA

B. Inactive DNA

                                                              B:A = 0:1          B:A = 20:1

   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).
A new and highly sensitive and selective Hg sensor

                Detection limit: 3 nM
                           Wang, Z; Lee, J.H; Lu, Y. Chem. Commun. 6005-6007 (2008)
“Dipstick” Litmus paper test for Pb2+ and other metals

                                                               It is now possible for even
                                                               simpler detection of
                                                               radionuclides and metal ions

  Debapriya Mazumdar, Juewen Liu, Geng Lu, Juanzuo Zhou and Yi Lu, Chem. Commun. 46, 1416-1418 (2010).
Sensing and “budgeted” release of chelators
         for on-demand metal ion remediation

M. Veysel Yigit, A. Mishra, R. Tong, J. Cheng, G. C. L. Wong, and Y. Lu, Chem. Biol. 16, 937 – 942 (2009).
Sensor product development

              www.ANDalyze.com
First test of ground water samples at ORFRC

                Area 3

                                      Thanks to
                                    David B. Watson
                                 Marcella (Sally) Mueller
                                   Kenneth A. Lowe
       Area 4                       N. Diane Kosier
                                   Tonia L. Mehlhorn
                                   Jennifer E. Earles
                                  Jesse M. Stephens
First test at ORFRC

                      Sensor

                       Hannah Ihms
Fundamental understanding of selectivity:
                 Using single molecule FRET

Hee-Kyung Kim, Ivan Rasnik, Juewen Liu, Taekjip Ha, and Yi Lu, Nature Chem. Biol. 3, 763-768 (2007).
Fundamental understanding of selectivity:
         Using biochemical assay and x-ray absorption
                        spectroscopy

Andrea K. Brown, Juewen Liu, Ying He, and Yi Lu, ChemBioChem 10, 486-492 (2009).
Bruce Ravel, Scott C. Slimmer, Xiangli Meng, Gerard C. L. Wong, and Yi Lu, Rad. Phys. Chem. 78, S75–79 (2009).
Future directions
Applications: New sensors: methylHg; Cr(VI), Pu(IV), Fe(II)/Fe(III)
             DNAzyme microarrays for multiplexing detection
                                                  Pb2+ UO22+

                                                               -, -
                                                               -, +
                                                               +, -
                                                               +, +

Fundamental Science: Elucidate structural features for selectivity

              UO22+ sensor       Pb2+ sensor
                (11 ppt)          (0.2 ppb)
Summary
 To obtain sensitive and selective biosensors, we have demonstrated the
following general strategies:
        • to obtain sensing molecules;
        • to improve selectivity;
        • to convert molecular recognition event into physically
        detectable signals (e.g., fluorescence and colorimetric);
        • to tune the dynamic range.
The new catalytic DNA fluorescent and colorimetric sensors
 environmentally benign
 cost effective
 stable under rather harsh conditions
 can be denatured and renatured many times
 allow combinatorial search for desired metal-binding properties
 adaptable to optic fiber and microarray chip technology
 unlimited by the choice of fluorophore/optical tags
 easy to attach fluorophore/optical tags to any desired position
 activity-based detection immune to source fluctuation and electronic drift
 highly sensitive (down to ppt) and selective (more than 1 million fold)
 allows on-site, real-time detection and quantification with high spatial (< cm)
and time (< min.) resolution.
 detect not only different metal ions, but also different oxidation states of the
same metal ions, allowing sensing of bioavailable toxic metal ions.
Acknowledgments
    DNA/RNA Lab at UIUC                          IFRC, ONRL
        Graduate Students:
                                                David B. Watson
Ying He        Andrea K. Brown
                                             Marcella (Sally) Mueller
Hannah Ihms Peter J. Bruesehoff
Tian Lan       Hee-Kyung Kim                   Kenneth A. Lowe
Darius Brown Jing Li                            N. Diane Kosier
Nandini Nagraj Jung Heon Lee                   Tonia L. Mehlhorn
Eric Null      Juewen Liu                      Jennifer E. Earles
Zidong Wang Kevin E. Nelson                   Jesse M. Stephens
Brian Wong     Daryl P. Wernette
Weichen Xu     Mehmet V. Yigit              Oregon State University
Hang Xing      Debapriya Mazumdar
                                               Jonathan D. Istok
Yu Xiang       Seyed-Fakhreddin Torabi
                                               Mandy Michalsen
        Postdoctoral fellows:
Hui Wei       Zehui Cao                            Funding
Juanzuo Zhou Geng Lu
                                               Department of Energy
                                         Office of Science, ERSP program
              Xiangli Meng
                                              (DE-FG02-08ER64568)
              Daisuke Miyoshi
              Wenchao Zheng
A sensor that resists to temperature-dependent variations

  Nandini Nagraj, Juewen Liu, Stephanie Sterling, Jenny Wu and Yi Lu, Chem. Comm. 2009, 4103-5.

    Label-free DNA sensors for cost-effective sensing

         Yu Xiang, Aijun Tong and Yi Lu, J. Am. Chem. Soc. 131,15352-15357 (2009).
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