Nanobiosensors for Nanobiosensors for Intracellular AnalysisIntracellular Analysis

Y. Zhang, P. Kasili, G. D. Griffin, and T. VoY. Zhang, P. Kasili, G. D. Griffin, and T. Vo--Dinh*Dinh*

Fitzpatrick Institute for Photonics Fitzpatrick Institute for Photonics Departments of Biomedical Engineering and Chemistry Departments of Biomedical Engineering and Chemistry

Duke UniversityDuke University

IntroductionIntroductionMany of the subcellular architectural features of the cell are on the scale of tens or hundreds of nanometers in size. Thus nanometer-sized probes can potentially be used to interrogate cellular activity in highly localized regions of the cell.

The optical nanosensor is able to interrogate a very localized area in the immediate vicinity of the probe’s tip (~ 100 nm) due to the constraints of the evanescent field excitation associated with the nanometer-scale tip, This nanosensor thus achieves an important requirement for intracellular probe design.

Schematic of animal cells(Ref:http://www.animalport.com/img

/Animal-Cell.jpg)

Mode propagation in a tapered metal-coated optical fiber

Silver

fiber

Evanescent decay

IntroductionIntroductionFiberFiber--optic nanobiosensors are optical optic nanobiosensors are optical nanoprobes which have biorecognition nanoprobes which have biorecognition molecules (i.e. antibodies, peptides) immobilized molecules (i.e. antibodies, peptides) immobilized on their tips.on their tips.When analyte molecules (proteins) are bound by When analyte molecules (proteins) are bound by the biorecognition molecules, changes in the the biorecognition molecules, changes in the fluorescence properties of light can be detected.fluorescence properties of light can be detected.Fiber optic nanosensor technology has the Fiber optic nanosensor technology has the potential to produce a new generation of potential to produce a new generation of nanobiosensors able to detect specific protein nanobiosensors able to detect specific protein targets at the singletargets at the single--cell levels.cell levels.

Nanoprobe for early detection of Nanoprobe for early detection of biomarkers in breast cancerbiomarkers in breast cancer

Development of breast cancer is thought to be a Development of breast cancer is thought to be a multimulti--step process resulting from accumulation step process resulting from accumulation of cellular damage. of cellular damage. Signal transduction pathways and transcription Signal transduction pathways and transcription factors have become attractive targets of factors have become attractive targets of chemoprevention, given their roles in breast cell chemoprevention, given their roles in breast cell growth and their responses to steroid hormones. growth and their responses to steroid hormones. Improved understanding of how cell signaling in Improved understanding of how cell signaling in the breast is altered could facilitate the the breast is altered could facilitate the development of more satisfactory preventive development of more satisfactory preventive agents and risk assessment strategies.agents and risk assessment strategies.

Nanosensor for caspase detectionNanosensor for caspase detection

Nanosensor has been developed for monitoring the Nanosensor has been developed for monitoring the onset of the mitochondrial pathway of apoptosis by onset of the mitochondrial pathway of apoptosis by detecting enzymatic activities of an indicator caspase.detecting enzymatic activities of an indicator caspase.Photodynamic therapy (PDT) protocols employing Photodynamic therapy (PDT) protocols employing δδ--aminolevulinicaminolevulinic acid (5acid (5--ALA) are an established means of ALA) are an established means of inducing apoptosis in MCFinducing apoptosis in MCF--7 cells.7 cells.LEHDLEHD--AMC as a caspaseAMC as a caspase--9 substrate was covalently 9 substrate was covalently attached to the nanosensor. Free AMC was generated attached to the nanosensor. Free AMC was generated by cleaving the substrate during apoptosis. By by cleaving the substrate during apoptosis. By quantitatively monitoring fluorescence signals, caspasequantitatively monitoring fluorescence signals, caspase--9 activity within a single living MCF9 activity within a single living MCF--7 cell was detected.7 cell was detected.

Nanoprobe fabricationNanoprobe fabrication

laser-pulled nanotip

Silver coated nanotip

Thickness monitor

Fiber probe

MotorTarget

Laser Mirror

Lens

PullerFiber

Laser-based fiber pulling

Angled evaporation

Nanoaperture fabricated by angled evaporation

B

NanoprobeNanoprobe fabrication

Nanoaperture fabricated by focused ion beam (FIB)

Functionalization of nanoprobeFunctionalization of nanoprobe

Amino group

Carboxyl group

Intermediate

Caspase substrate

NH2

SiOEtEtO

OEt

O O O

SiO2OOO

Si NH

OH

O O

EDC/NHS

SiO2OOO

Si NH

O

O ON

O

O

Caspase substrateSiO2

OOO

Si NH

LEHD-AMC

O O

SiO2OOO

Si NH2SiO2

OHOHOH

Diagrammatic representation of 5-ALA induced apoptosis, involving the activation of caspase-9 followed by the cleavage of LEHD-AMC, and subsequent detection of free AMC

5-ALA induced apoptosis

Caspase activation

LEHD AMC

LEHD AMC

LEHD

λex=325nm

AMC

λem=440nm

350 400 450 500 5500

1x106

2x106

3x106

4x106

5x106

Inte

nsity

(cou

nts)

Wavelength (nm)

NucleusMitochondria

Apaf1

Inactivate procaspase-9

Permeable Mitochondria

Active caspase-9

5ALA λex=632.8nm

Apoptosis induction

MCF-7 cell Apoptotic MCF-7 cell

Bcl-2

Cytochrome C

Figure 6. Phase contrast image of a nanosensor

inside a living cell.

Figure 5. Schematic diagram of fluorescence measurement system

Nanosensor detection in single cellNanosensor detection in single cell

CaspaseCaspase--9 activity in living cell 9 activity in living cell

(A) The background-corrected results of intracellular detection of caspase-9 activity in experimental group of MCF-7 cells using six replicate nanosensors, one for each measurement.

(B) The background-corrected results of a series of three intracellular measurements performed with a nanosensor inserted into single live MCF-7 cells of the treated control group.

(A) (B)

In vivo measurement of CaspaseIn vivo measurement of Caspase--9 9 activityactivity

0

15000

30000

45000

60000

75000

Fluo

rese

nce

Inte

nsity

(RFU

)

Group I: (+)ALA (+)PDT Group II: (+)ALA (-)PDT Group III: (-)ALA (+)PDT Group IV: (-)ALA (-)PDT

0

15000

30000

45000

60000

75000

Fluo

rese

nce

Inte

nsity

(RFU

)

Group I: (+)ALA (+)PDT Group II: (+)ALA (-)PDT Group III: (-)ALA (+)PDT Group IV: (-)ALA (-)PDT

ConclusionConclusionFiber opticFiber optic--based nanosensors based nanosensors have been have been utilized to study individual cells without having to utilized to study individual cells without having to disrupt their physiological makeup, which in the disrupt their physiological makeup, which in the process can negatively interfere with cellular process can negatively interfere with cellular biochemistry.biochemistry.These These nanodevicesnanodevices could also be used for could also be used for advanced biosensing systems in order to study advanced biosensing systems in order to study in situ intracellular signaling processes.in situ intracellular signaling processes.Nanobiosensor will lead to significant advances Nanobiosensor will lead to significant advances in the diagnosis and prevention of disease at the in the diagnosis and prevention of disease at the molecular level.molecular level.

Future workFuture work

Carry out similar analysis on other proteins Carry out similar analysis on other proteins involved in biochemical cellular pathwaysinvolved in biochemical cellular pathwaysEExplore the sensitivity and selectivity of xplore the sensitivity and selectivity of multiplexed protein biomarker detection multiplexed protein biomarker detection using an antibodyusing an antibody--based immunoassaybased immunoassayDevelop Develop plasmonicsplasmonics--enhanced enhanced nanoprobes for DNA sensing by fabricating nanoprobes for DNA sensing by fabricating metallic nanostructures on fiber optic tipsmetallic nanostructures on fiber optic tips

ReferenceReference[1] Y. Zhang, H.-N. Wang, M. Gregas, P.M. Kasili and T. Vo-Dinh, “Nanobiosensors for analysis of single living cells,” Fitzpatrick Institute for Photonics 7th Annual Meeting, Oct 2007.[2] T. Vo-Dinh, P. Kasili, and M. Wabuyele, "Nanoprobes and nanobiosensors for monitoring and imaging individual living cells," Nanomedicine, vol. 2, pp. 22-30, Mar 2006.[3] T. Vo-Dinh and P. Kasili, "Fiber-optic nanosensors for single-cell monitoring," Analytical and Bioanalytical Chemistry, vol. 382, pp. 918-925, Jun 2005.[4] P. M. Kasili and T. Vo-Dinh, "Optical nanobiosensor for monitoring an apoptotic signaling process in a single living cell following photodynamic therapy," Journal of Nanoscience and Nanotechnology, vol. 5, pp. 2057-2062, Dec 2005.[5] P. M. Kasili, J. M. Song, and T. Vo-Dinh, "Optical sensor for the detection of caspase-9 activity in a single cell," Journal of the American Chemical Society, vol. 126, pp. 2799-2806, Mar 10 2004.[6] T. Vo-Dinh, "Nanobiosensors: Probing the sanctuary of individual living cells," Journal of Cellular Biochemistry, pp. 154-161, 2002.[7] T. Vo-Dinh, J. P. Alarie, B. M. Cullum, and G. D. Griffin, "Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell," Nature Biotechnology, vol. 18, pp. 764-767, Jul 2000.

Acknowledgement: This research was sponsored by the National Institute of Environmental Health Sciences (1R01ES014774)