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Ultrasensitive Nonlinear Multi-Photon Laser Wave-Mixing Detection Methods for Environmental and Biomedical Applications

Nonlinear multi-photon laser wave-mixing detection coupled with capillary electrophoresis is presented as ultrasensitive methods for a wide range of biomedical and environmental applications including simultaneous analysis of malachite green, crystal violet, their metabolites leuco-malachite green and leuco-crystal violet. Nonlinear wave mixing offers inherent advantages over conventional laser methods including zepto-mole detection sensitivity, excellent chemical selectivity and specificity levels, and high spatial resolution suitable for single-cell analyses. The wave-mixing signal is a coherent laser-like beam, and hence it can be collected with excellent signal-to-noise ratios and high detection efficiency levels. Chromatic and leuco forms of crystal violet and malachite green absorb in the UV and visible wavelength ranges. We use a 266 nm UV laser to probe label-free analytes in their native form and a visible laser to probe labeled analytes. The wave-mixing signal has a quadratic dependence on analyte concentration, and hence, wave mixing is especially effective for monitoring small changes in analyte properties. In order to further enhance chemical selectivity levels, a capillary (75 µm inside diameter) is used to flow and separate analytes in our custom-built capillary electrophoresis system. The wave-mixing probe volume is small (nL, pL), and hence, it is inherently suitable for interfacing to lab-on-a-chip, microfluidics and microarray systems. Excellent detection sensitivity levels (atto-mole to zepto-mole levels) have been demonstrated using capillary- and chip-based separation systems for different biomarkers and environmental samples. Our nonlinear multi-photon detectors can be easily configured as battery-powered portable devices that are suitable for use in the field where resources are limited. Hence, wave-mixing methods allow application in the field for a wide range of environmental and biomedical applications (biomarkers, viruses and early detection of cancer, etc.).

Introduc4on  

Experimental  Setup  

Results  

Limits  of  Detec4on  

1.  http://www.fao.org/3/contents/dbb1161a-8ac7-5128-94db-383e45cfcae3/AC343E00.HTM

2.  Romero, Jaime; Feijoo, Carmen Gloria; Navarrete, Paola. Antibiotics in Aquaculture – Use, Abuse and Alternatives. Institut for Nutrition and Food Technology.

3.  http://pubchem.ncbi.nlm.nih.gov/compound/Leucomalachite_green#section=Metabolism-Metabolites

Megan  Murphy,  Mya  Brown,  Jean  Sebas.en  Pradel  and  William  G.  Tong Department  of  Chemistry  and  Biochemistry,  San  Diego  State  University,  San  Diego,  CA  92182  william.tong@sdsu.edu  

Abstract  

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&=Λ

2sin2 θλ

I3 = Signal Beam Intensity I1 = Signal Beam Intensity I2 = Pump beam intensity from second laser beam b = Pathlength of laser beam cross section λ = Wavelength Θ = Angle between incoming beams dn = Change in index of refraction dT = Change in temperature α = Absorptivity Coefficient k = Thermal conductivity b = Path length

Laser Wave-Mixing Grating

Wave-Mixing Signal Variables  

DFWM Setup

•  Laser wave mixing can detect Malachite Green, Crystal Violet and their metabolites with 266 nm UV laser

References  

 Acknowledgments  

Support NIH (R01) NSF NIH NIGMS SDSU IMSD 2R25GM05898906-13 NSF HRD-1302873 ARCS Foundation U.S. Army Research Office U.S. Dept Defense (CCAT) U.S. Dept Homeland Security Johnson and Johnson Beckman CSUPERB

Advantages  of  Laser  Wave  Mixing    

TongLab (2014-15) Marcel Hetu (PhD) Manna Iwabuchi (PhD) Sebastian Pradel (PhD) Eric Maxwell (MS) Jeff Gilmour (MS) Alex Jackson (MS) Zarina Munshi (MS) Sashary Ramos (BS) Samer Gazale (BS) Kelsey Hunt (BA) Mya Brown (BS) Megan Murphy (BS)

•  MG and CV are triphenylmethane dyes •  Have carcinogenic and mutagenic properties •  Readily absorbed into fish tissue, •  Reduced metabolically to LMG and LCV •  MG and CV have been banned for use as fungicides and

antiseptics in aquacultures by the FDA and EU •  Necessary to develop sensitive, rapid, inexpensive and

reliable methods for the determination of CV, MG, LCV and LMG

Non-linear wave mixing spectroscopy is a detection to detect specific and sensitivity levels. Advantages of this method include: 1.  Enhanced sensitivity •  Quadratic dependence on an analyte 2. Small probe volume •  Fundamentally suitable for capillary electrophoresis 3. Not limited to fluorescing samples. •  Fluorophores and chromophores can be detected Laser wave mixing offers small probe volumes (nL) and it can be coupled with capillary electrophoresis and other capillary-based separation systems for separation and detection of environmental pollutants and proteins.

•  An.bacterial,  an.fungal  and  an.parasi.c  agent[3].  

•  Topical  an.microbial  for  treatment  of  human  skin  infec.ons[3].  

•  Ac.ve  in  oxidized  form  and  inac.vated  upon  reduc.on  or  decoloriza.on  to  leukomalachite  green[3].  

-­‐0.15  

0.05  

0.25  

0.45  

0.65  

0.85  

1.05  

1.25  

1.45  

240   250   260   270   280   290   300  

Absorbance  Units  

Wavelength  (nm)  LMG  1E-­‐3M   LCV  1E-­‐3M   MG  1E-­‐4M   CV  1E-­‐4M  

UV-Visible Spectrum of MG/LMG and CV/LCV

UV-­‐visible  spectrum  of    MG  and  CV  with  their  respec.ve  metabolites  that  indicates  266  nm  wavelength  of  the  UV  laser  used  in  the  wave-­‐mixing  setup.  Our  excita.on  wavelength  at  266  nm  is  close  to  the  λmax  for  all  four  analytes.  

-­‐0.01  

0.04  

0.09  

0.14  

0.19  

0.24  

0   10   20   30   40   50   60   70   80   90  

Intensity

 (V)  

Time  (s)  

5e-­‐8  M  Methyl  Red  in  Methanol  

Wave-mixing signal produced by 266 nm laser probing 5x10-8 M Methyl Red solution. The S/N ratio is 92:1. Methyl Red is used for optimizing optical alignment and S/N enhancement.

-­‐0.02  

0.03  

0.08  

0.13  

0.18  

0   20   40   60   80   100   120   140   160   180   200  

Intensity

 (V)  

Time  (s)  

LCV  Run  1  1e-­‐4   LCV  Run  2  1e-­‐4  

LMG  

MG  

LCV  

CV  

Mg/LMG and CV/LCV Detection using Ammoniumacetate Buffer in Capillary Electrophoresis

Electropherogram of MG/LMG and CV/LCV in 50 mM ammoniumacetate buffer (pH 4) obtained with in CE coupled with UV laser. Injection was done at 12kV,140 uA, for 3s, and 50 mM ammoniumacetate buffer (pH 4) was used as running buffer.

Cubic  dependence  on  laser  power  

 Square  dependence  on  absorp.vity