Silicon On-chip Open Bio-sensors with ultra-high Sensitivity requiring the smallest Sample Volume for Biomedical Diagnostics

Xtreme Sens Diagnostics Ray T. Chen1, Swapnajit Chakravarty2

Weicheng Lai1, Yi Zou1, Andreas Hsieh1, and Liang Zhu1

1 Microelectronics Research Center, Department of Electrical and Computer Engineering, University of Texas at Austin, 10100 Burnet Rd. Austin, TX 78758 USA

2 Omega Optics, Inc., 10306 Sausalito Dr., Austin, TX 78759 USA

Funded by National Cancer Institute SBIR Contract #: HHSN261201000085C, HHSN261201200043C

AFOSR MURI

Baylor College of Medicine, Houston, TX

Outline

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• Application in Lung Cancer Cell Line Lysate Detection

• Motivation • Photonic Crystal Sensor Introduction for Microarrays

• Detection Principle • Device Sensitivity

• Multiplexing • Specificity

• What we will demonstrate at our booth

• Directions for Future Collaboration and Research

Opal, the best known periodical structure in nature.

Photonic Crystal Structures in Nature and in Nanofabrication

3

Nanomembrane-based Open Sensor Systems on Silicon PCW Chip

Early Cancer Detection (NIH)*

Air-pollution (Methane gas) Sensor(EPA)*

Water-pollution (Xylene) Sensor (NSF)*

Sensor for National Security (Chemical Warfare Simulant TEP Triethyl-phosphate, funded by US Army)*

EM-Wave Sensor (Missile Defense Agency)*

*: Peer-reviewed Journal publications

available or to be available

What has been demonstrated

On Semiconductor Nanomembranes

5

The Sensor on a System Level

PC Microcavity

Group Index Taper

Sub-Wavelength Grating Coupler

6

Principle of Operation

Wavelength (nm)

Inte

nsity

(a.u

.)

Wavelength (nm)

Inte

nsity

(a.u

.)

7

Principle of Operation

Wavelength (nm)

Inte

nsity

(a.u

.)

Wavelength (nm)

Inte

nsity

(a.u

.)

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Principle of Operation

Wavelength (nm)

Inte

nsity

(a.u

.)

Wavelength (nm)

Inte

nsity

(a.u

.)

Association Rate Ra = ka [A] (1-[AB])NB

[A] [B] ↔[AB] [B]: Target Protein Concentration [A]: Probe Protein Concentration NB: Number of Target molecules on the sensor surface

Dissociation Rate Rd = kd [AB]NB Dissociation Rate Constant: kd Association Rate Constant: ka

[AB]: Fraction of surface molecules occupied by a probe molecule

Equilibrium is reached when kd = ka

Binding Mechanism

nA: Refractive Index of Adsorbed Molecules

nC: Refractive Index of PBS

dn/dc: Change in refractive index versus change in thickness of adsorbed layer

dA: Thickness of Probe Protein Layer

dl: Resonance Wavelength Shift

De Feijter’s Formula:

10

Binding Measurements

Lai et al. Optics Lett. 37, 1208 (2012)

Functionalization:

• 10% by volume 3-APTES in toluene.

• washed 3 × in toluene to remove

unbound 3-APTES, 3 × in

methanol to remove toluene and 3 ×

in de-ionized water to remove

methanol.

• wafers incubated in 1%

glutaraldehyde in PBS for 5 minutes

• washed 3 × in PBS and ink-jet

printed with target antibodies in 35%

glycerol.

• printed spots incubated overnight at

4oC.

• washed 3 × in PBS

Probe Protein Binding

Target Protein Binding

Sensitivity Comparison

Zou et al., (in review)

Sensitivity Comparison

At 60fM, ring resonator has wavelength shift of 0.2pm at 1550nm

At 50fM, photonic crystal sensor has wavelength shift of 50pm at 1550nm

Photonic crystal sensor is 250 times more sensitive than ring resonator

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• CMOS platform--- low cost during high volume manufacturing

• High sensitivity demonstrated at 0.1mg/ml

• 50fM concentration of avidin binding to biotin detected

• High “working device” yield of photonic crystal microcavity biosensors using sub-wavelength grating couplers

• Repeatable resonance wavelength shifts

• Multiplexed Devices for Microarrays

Application in Lung Cancer Detection with Sensitivity and Specificity

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Lung Cancer Cell Lysates

• NCI-H358 lung cancer cell line stably transfected with a tetracycline-inducible 6myc-ZEB1 expression construct

• Samples were prepared from these cells prior to induction (0-day lysate) or after 3 days dox treatment to express 6myc-ZEB1 (3-day lysate).

• Exogenous 6myc-ZEB1 present in the induced lysates can be detected using either the 9E10 antibody or an antibody that binds to native ZEB1

Specific Antibodies: • Anti-ZEB1 • Anti-MYC 9E10

Specificity by Sandwich method

• Sample fairly representative of complex media, since ZEB1-infected cells must be detected from a mixture of 20K-30K proteins.

Multiplexed Sensitivity and Specificity

4 PC Microcavities in Silicon

Immobilized Antibodies

3. Anti-ZEB1 4. Anti-MYC 9E10 2. Mouse IgG 1. BSA

Induced Lysates

Induced Lysates to attach to ZEB-1 and MYC 9E10

ZEB

MYC 9E10

1 2 3 4

1 2

3 4

Chakravarty et al., Biosensors and Bioelectronics 43, 50 (2013)

Multiplexed Sensitivity and Specificity

Immobilized Antibodies

3. Anti-ZEB1 4. Anti-MYC 9E10 2. Mouse IgG 1. BSA

Induced Lysates

Induced Lysates to attach to ZEB-1 and MYC 9E10

ZEB

MYC 9E10

Washing in PBS

Mouse IgG1

4 PC Microcavities in Silicon

l3 Shift l4 Shift l1 No Shift l2 No Shift

1 2

3 4

1 2

3 4

Chakravarty et al., Biosensors and Bioelectronics 43, 50 (2013)

Multiplexed Sensitivity and Specificity

3. Anti-ZEB1 4. Anti-MYC 9E10 2. Mouse IgG 1. BSA

Induced Lysates to attach to ZEB-1 and MYC 9E10

ZEB

MYC 9E10

Mouse IgG1 Washing in PBS

Anti-MYC 9E10

l3 No Shift l4 No Shift l1 No Shift l2 No Shift

1 2

3 4

Chakravarty et al., Biosensors and Bioelectronics 43, 50 (2013)

Multiplexed Sensitivity and Specificity

Chakravarty et al., Biosensors and Bioelectronics 43, 50 (2013)

3. Anti-ZEB1 4. Anti-MYC 9E10 2. Mouse IgG 1. BSA

Induced Lysates to attach to ZEB-1 and MYC 9E10

ZEB

MYC 9E10

Sandwich for Specificity l3 Super

Shift

Washing in PBS Anti-MYC 9E10

1 2

3 4

• Initial Cell Concentration: 10,000 cells per micro-liter made up of 20K-30K proteins

Sensitivity Amplification by Sandwich Assay

• Samples diluted in PBS: 60 micro-liters in each addition

• 10 cells per micro-liter detected without sandwich

• 2 cells per micro-liter detected with sandwich assisted amplification

Chakravarty et al., Biosensors and Bioelectronics 43, 50 (2013)

1 2 10 100 1000 5000 0d T2 Cells per micro-liter

2 1 ZEB1 detected from among 20K-30K proteins

What we Will Demonstrate

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Biomolecules need better sample control than what we can demonstrate here at the booth in a quick experiment • Basic principle of operation of device will be demonstrated • Target biomolecules cause change in refractive index • Instead of 2 different concentrations of target biomolecules, we

will demonstrate the principle with two different target analytes, water and sugar solution.

Raw Spectrum In Water In Sugar Solution

What we Seek

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• Early Detection, Signaling Pathway Identification

• Partnerships / Joint R&D for New Applications, New Products

• Medical Research and Drug Discovery

• Cancers, Infectious Diseases

• Other Pathogens….Environmental Sensing, Food and Beverages, Ion Sensing in Solvents, Bio-Warfare Agents

• Extendable to nucleic acids, small molecules etc……Gene Sequencing

• Long Term: Screening

Research Avenues (NIH)

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• R01-type (NIH) • NIAID: RFA-AI-13-013: Partnerships for Biodefense

• NIAMS: PA-12-191, RFA-AR-14-005: Multiplex Assay Development for Arthritis and Musculoskeletal and Skin Diseases

• NHLBI: HLS13-13: Innovative Point-of-Care technologies to guide diagnostic, monitoring or therapeutic efforts in clinical studies of cardiovascular, lung, and blood diseases and disorders • NHLBI: HLS13-03: Diagnostics: devices, biomarkers, imaging, and assays for non-malignant blood disorders

• SBIR Omnibus (NIH)

• R43-type (NIH)

• R21/ R33-type (NIH) • NIAID: RFA-AI-13-019: Drug Target Development and Validation for Antimicrobial-Resistant

Pathogens • NCI: RFA-CA-13-001, RFA-CA-13-002: Early-Stage Innovative Molecular Analysis Technology

Development for Cancer Research

Research Avenues (DoD)

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• DoD • DARPA: SB132-001 Improving Measurement Sensitivity and Specificity of Oxytocin

• Oxytocin: reproduction, childbirth, neuromodulator

• ARMY: A13-082: Rapid Point of Care Therapeutic Drug Monitoring Device • peak and trough concentrations of antibiotics and antifungals in blood and serum

• Simultaneous detection of proteins and nucleic acids

• NSF

• New ERC Proposal preparation for Open sensing Systems

Thank You

Please Visit Us at Our Booth

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• Only 60 micro-liters of sample used for 4 simultaneous binding measurements…..translates to low patient sample requirement

• Photonic Crystal microcavity biosensors in silicon have shown highest sensitivity at 0.1mg/ml with high yields (95%)

• Lung Cancer Cell Lysates detected to concentrations of 2 cells per micro-liter in a mixture of 20K-30K proteins

• Label-Free Specificity demonstrated by multiplexing and sandwich assays…..translates to lower false positives

• Multiplexed Simultaneous binding measurement at same instant of time……..translates to high throughput diagnostics

• Simultaneous Detection of Multiple Diseases….translates to low cost of ownership for medical facilities, reduced cost for patient

L13 PhC microcavity structure

PhC one each arm

Multiplexing Sensors for Simultaneous Detection

Zou et al. , Lab Chip 12, 2309 (2012)

Device Yield (95% for L13 across 3 different chips)

(a)

Zou et al., (in review)

Device Measurement Repeatability

Zou et al., (in review)

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Band Engineering for Efficient Coupling in Slow Light Guiding Wavelengths

Group Index Taper Design by Hole Radius Gradient

Lin et al. , Appl. Phys. Lett. 97(18), 183302 (2010)

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Bio-Sensing

Target Antibody Probe Antibody Kd (M) Dissociation Constant

Rabbit anti-goat IgG Goat anti-rabbit IgG ~10-6 (Kuo et al., 1993) Human IL-10 IL-10, Rat Anti-Human ~10-9 -10-11 (de Groote et al, 1994)

Biotin Avidin ~10-15 (Scullion et al., 2011)

Chakravarty et al., Biosensors and Bioelectronics 38(1), 170 (2012)

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Microcavity Engineering for High Sensitivity

PC Microcavities in SOI devices Experimental Q~26,760 in IPA

Lai et al. Optics Lett. 37, 1208 (2012)

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Factors in microcavity integration in a microarray

• Ink-Jet Dispensed Spot Size

• Minimum distance between unique ink-jet printed spots

Biomolecule Patterning

Minimum ink-jet dispensed spot: 35mm Minimum distance between ink-jet dispensed spots: 50mm

Lai et al. Optics Lett. 37, 1208 (2012)

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Microcavity Engineering for High Sensitivity

Quality factor of photonic crystal microcavities

QR=wtR ; tR represents the radiation loss and Qi=wti, ti represents the intrinsic cavity loss

PR = total power radiated by the cavity ↓ WE = stored energy in the cavity which is proportional to the cavity mode volume ↑

Chakravarty et al. Biosens. Bioelectron. 38(1), 170 (2012)

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Multiplexed Detection #1-#3: Single photonic crystal microcavities in separate waveguides #4: Two photonic crystal microcavities in a single waveguide All waveguides excited at the same time

#1: Coated with Human IL-10

#3: Coated with Rabbit Anti-Goat IgG

#3: Coated with BSA

#4: Rabbit Anti-Goat IgG and BSA

Zou et al. , Lab Chip 12, 2309 (2012)

1

3

2

4

• Resonance Wavelength Shift with Induced Lysate in PBS. • Specificity demonstrated further with

isotype matched control mouse IgG1

Specificity

• No Resonance Wavelength Shift with Un-Induced Lysate in PBS.

• Secondary Supershift with secondary antibody in PBS: Sandwich

Specificity is achieved with Label-free Sandwich Assays

• Secondary Supershift with secondary antibody in PBS: Sandwich

Chakravarty et al., Biosensors and Bioelectronics 43, 50 (2013)