2. 6x10 ° -
2 5x107
E
;C
4x10
U
U
4E. 3x10
d
O
U
o
2x10
:4=
U
; 1x10
x
w
-Glycerophospholipid
-Germanium with AR
coating
-0.6
3
A,
5 6 7
Wavelength (µm)
8 9
0.5
0.4 `c3
C
ß
..-.
0.3 E
Cl)
C
co
0.2 i-
0.1
0.0
10
2. LABEL FREE DETECTION
An efficient identification of a specific biomarker in solution using a lab-on-chip platform requires minimal sample
preparation and labeling, with the capability of being integrated into single low cost platform. Also, it is important to
identify many unique biomarkers from the same sample. However, at present the most common label-free approach,
mass spectrometry, couples a micro-scale chip with an instrument that is ≥100x the size of the chip [3, 4, 5].
Lab-on-chip (LOC) diagnostic technology has provided low cost, compact diagnostic platforms useful in both
advanced clinical settings and the developing world. These devices leverage semiconductor fabrication techniques to
interact small volumes of analyte with electrical, optical, or chemosensitive sensors. It is not easy to distinguish
many analytes without the introduction of an extrinsic contrast agent (e.g., immunohistopathological labels or
selective dyes) or functionalized detection surfaces. But, these steps add significant cost and complexity, which
eliminates some of the most important advantages of the LOC platform. As a result, it is crucial to develop and
characterize label-free LOC techniques for the advancement of the technology.
3. DETECTION OF PHOSPHOLIPIDS IN A NONAQUEOUS SOLVENT BY MIR
ABSORPTION
We intend to use the mid-IR determination of phospholipids in the non-aqueous solvent N-methylformamide
(NMF). We use non-aqueous solvent to overcome the presence of strong water absorption, the principle limitation of
the MIR detection. Phospholipids exhibit strong MIR absorption peaks, as tabulated in Table 1. Fortunately, the
strong characteristic vibrational bands associated with symmetric and asymmetric phosphate stretching, ν(PO2-),
occur in regions of the fingerprint region where the solvent absorption is relatively weak, exhibiting only 3 minor
bands over the 1000-1300 cm-1
region. A particularly important class of phoshpolipids is glycerophospholipids,
which are derived by various metabolic pathways from phosphatidic acid (PA) as a base molecule [7, 8]. Therefore,
molecules like phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE),
phosphatidylglycerol (PG), and phosphatidylserine (PS) will all have a similar structure to PA. To evaluate the
feasibility of MIR absorption of glycerophospholipids, we measured the MIR absorption of a 12-carbon chain PA
(1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt)) dissolved in NMF, which, as expected exhibits a strong amide
peak near 1660 cm-1
(6 μm), arising from the NMF solvent, and the phosphate headgroup vibrations of the model
analyte (Figure 1).
Figure 1. MIR absorption of a 12-carbon chain PA dissolved in NMF and the MIR transmission through a Ge sample with Si3N4
anti-reflection coating.
Proc. of SPIE Vol. 9166 91660H-2
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3. Malyte
Reservoir
Separation
Micro-channel
Butler
Sample
Buffer Buffer
Sample
ii ii
Table 1. Common phospholipids MIR absorption bands [9, 10, 11, 12].
Annotated Functional Group Wavenumber (cm-1
)
νs(CHx), νas(CHx) 2851, 2872, 2920, 2960, 3010
γ(CH2) 1470
γs(CH3) 1380
w(CH2) 1340-1370
νas(PO2
-
) 1240
νs(PO2
-
) 1090
δ(CH2) 720
ν = stretching γ = bending w = wagging δ = rocking s = symmetric as = antisymmetric
4. MICROCHIP ELECTROPHORESIS
The planar microchip electrophoresis device consists of a sample channel and a separation channel that intersect in a
“cross” fashion. A pinched injection strategy is used to dispense discrete sample volumes at the cross intersection
into the separation channel for transport and separation and downstream detection [6]. To determine the proper
voltage scheme, a hydraulic/electrical circuit model was used to calculate pressure/flow rate and voltage/current
values in the microfluidic channels. Microfluidic chips were fabricated using standard photolithography and soft
lithography. Briefly, SU-8 photoresist was spin-coated on 4 inch silicon wafers which were then soft-baked,
photopatterned, hard-baked, and developed to produce microscale SU-8 features. Polydimethylsiloxane (PDMS)
prepolymer was then poured over the SU-8/silicon mold and cured in an 80 degrees Celsius oven for two hours. The
microdevice is then lifted off of the mold and bonded to a germanium wafer. Using this approach, we tried to use
very thin PDMS layer to host the microfluidic channel as the MIR absorption through the PDMS increase with its
thickness.
Figure 2. Different steps of a capillary electrophoresis.
5. STUDY ON THE MIR DETECTION SYSTEM DESIGN
To study the feasibility of on-chip mid-IR detection for phospholipids, we propose to use a very simple structure
comprising of Ge substrate holding a PDMS microfluidic channel. Ge shows good transmission in mid-IR, and also
cheap compared to the other mid-IR detection platform material such as CaF2 or ZnSe [13]. To measure the mid-IR
transmission characteristics of Ge we used a FTIR system. The FTIR spectrum of a bare Ge sample showed around
38% transmittance at a wavelength of 6 µm. This is the wavelength corresponding to the dominating peak of the
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4. Wavelength (µm)
10 9 8 7 6 5 4 3
1.0 1 ,
-PDMS/Ge (200 pm thick PDMS)
-Ge spectrum (470 pm thick)
-PDMS spectrum (400 pm thick)
0.8 - tff titi/-tt
0.6 -
0
U -
C
w 0.4-
E
o
c
0.2-
0.0
1000
, , , ,
1500 2000 2500 3000 3500
Wavenumber (cm-1)
phospholipid extinction co-efficient in our previous ATR measurement (Figure. 1). On-chip electrophoresis requires
an insulating layer on the Ge substrate. Also, the transmission characteristics through the Ge can be engineered with
the introduction of an anti-reflection (AR) coating on top of the Ge surface. We used a PECVD Si3N4 layer as the
AR coating. A Si3N4 AR coating of 1050 nm (~λ/2n of 6 µm wavelength, where λ is the wavelength and n is the
refractive index=~1.8), we found a higher transmission window around 6 µm wavelength (Figure. 1). So, the
detection sensitivity is increased by improving the Ge transmission characteristics with the use of an AR coating.
Next, we performed experiments to see the feasibility of using a PDMS micro-fluidic channel on top of the Ge
substrate. The goal of the experiment was to figure out how transmission through the PDMS layer varies with
thickness. We did an ATR measurement of a PDMS sample. From the ATR measurement data the absorption co-
efficient of PDMS is calculated as 35.5 cm-1
at the 6 µm wavelength. This value corresponds to a 20% transmission
through a 500 µm thick PDMS layer. Experimentally, we were able to make a thin PDMS layer of 200 µm with a
50 µm high microflidic channel within that. The FTIR transmission data for the PDMS/Ge sample is shown in
Figure 3. Our experimental lead us to design our microfluidic channel using a very thin PDMS channel on a Ge
substrate. To make the channel more stable, we put a thick PDMS layer of around 1 cm, and cut a window where the
mid-IR light is intended to pass through. An image of the fabricated PDMS/Ge structure is given in Figure. 4.
Figure 3. Mid-IR transmission through PDMS/Ge microfluidic structure.
Figure 4. Fabricated PDMS/Ge microfluidic structure with measurement window.
Proc. of SPIE Vol. 9166 91660H-4
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5. 0.1
.01
1E-4 1 1 1 1 1 I 1 1
4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Wavelength (µm)
To determine the detection sensitivity for this structure, the mid-IR transmission through the 200 µm PDMS/Ge
sample with a 50 µm high microflidic channel within. With measure the standard deviation of the measurement,
Using Beer-Lambert law we measured the corresponding phospholipids concentration-length product for this design.
According to the Beer-Lambert law,
So,
Where, = input signal,
= thickness of the channel
= extinction co-efficient
and = molar concentration
Now,
So,
And considering the scan integration time (T=1.679 s),
So, = (3)
The measured concentration-length product for our experiment is plotted in Figure 5. The result is summarized in
Table. 2:
Figure 5. Concentration-length product with wavelength variation.
Table 2. Concentration-length product lower peak values:
Wavelength (µm) Extinction co-efficient, Concentration-length
product, (μm M Hz-1/2
)
4.2 .035
6 .0007
6.5 .005
7.25 .0005
Proc. of SPIE Vol. 9166 91660H-5
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6. From the graph plotted in Figure 5. the minimum detectable concentration calculated for 6 μm wavelength IR light
passing through a 50 μm thick channel is found to be 140 μM. This value is higher than the phospholipid present in
human body fluid as biomarkers. So, to measure phospholipid by using mid-IR we need to increase the integration
time for the measurement, that is not compatible with the electrophoresis separation. So, we need to introduce
compact sensing with mid-IR source coupled with the detection platform [14, 15]. Also, the light needs to be
directly coupled to the waveguide which should be put across the microfluidic channel. So, an effective high-
efficiency optical coupling technique could provide better platform for mid-IR phospholipid sensing as well [16,
17]. Applying this compact sensing technique the noise due to atmospheric vapor and CO2 can be minimized. As a
result, the technique has the potential to decrease the integration time during scanning to make mid-IR label free
detection compatible with electrophoresis separation.
6. CONCLUSION
Mid-IR label free sensing is a promising technique for building the on-chip compact sensor for phospholipid
biomarker detection. Our study concentrates on finding the requirements and feasibility of this technique. The future
work would combine both fabricating the on-chip platform to do the sensing and improving the chip-to-chip optical
coupling to enhance the detection sensitivity.
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