We describe an automated, self-powered chip based on lateral flow immunoassay for rapid, quantitative, and multiplex protein detection from pinpricks of whole blood. The device incorporates on-chip purification of blood plasma by employing inertial forces to focus blood cells away from the assay surface, where plasma proteins are captured and detected on antibody “barcode” arrays. Power is supplied from the capillary action of a piece of adsorbent paper, and sequentially drives, over a 40 minute period, the four steps required to capture serum proteins and then develop a multiplex immunoassay. An 11 protein panel is assayed from whole blood, with high sensitivity and high reproducibility. This inexpensive, self-contained, and easy to operate chip provides a useful platform for point-of-care diagnoses, particularly in resource-limited settings

Ahmad, Habib

Heath, James

Ma, Chao

Shi, Qihui

Vermesh, Ophir

Vermesh, Udi

Wang, Jun

Caltech Authors - Main

Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
Electronic Supplementary Information (ESI) 
 
A Self-powered, One-step Chip for Rapid, Quantitative and Multiplex 
Detection of Proteins from Pin-pricks of Whole Blood 
Jun Wang, Habib Ahmad, Chao Ma, Qihui Shi, Ophir Vermesh, Udi Vermesh & James Heath* 
NanoSystems Biology Cancer Center, Kavli Nanoscience Institute, Division of Chemistry and 
Chemical Engineering, California Institute of Technology, MC 127-72, 1200 E. California Blvd., 
Pasadena, California, 91125, USA.   
 
*Correspondence should be addressed to J.R.H. (heath@caltech.edu). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
 
Supplementary Figures 
 
Supplementary Figure 1. Design of the LF-IBBC chip. The layout includes two layers, layer 1 
for fluidic flow and layer 2 for filter paper insertion. All the chambers are connected by an array 
of posts which function as a filter to remove impurities and as a flow resistor. The minimum 
distance between posts in the last post array before the serpentine channel is designed to be 10 
µm, which effectively dams large impurities and prevents clogging of the narrow, 12 µm channel 
ahead. The serpentine channel incorporates Dean vortices at each turn to mix solutions. The 
following narrow channel can effectively focus blood cells and separate them from plasma. 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
 
Supplementary Figure 2. Profile of flowing blood cells at barcode assay channel as a 
consequence of focusing at the narrow channel. (a) Effect of narrow channel width on cell 
focusing profile. (b) Effect of flow velocity on focusing profile when the narrow channel width 
was 12 µm. In the range between 0.25 µl/min and 4 µl/min, the flow velocity has negligible 
effect on blood cell focusing. The blood cells tend to be nonspecifically captured by stripes as 
well as the channel’s bottom surface when the velocity is less than 0.5 µl/min.  
 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
 
Supplementary Figure 3. Fabrication of DNA barcode array. High-density channels guide 
DNA oligomers to form a compact DNA array with as many as 36 replications shown in (a). The 
flow patterning is accomplished via the microfluidic device illustrated in (b), which is comprised 
of a PDMS replica bonded to a polylysine glass slide to seal the channels. Prior to conducting 
protein experiments, a section of each slide is tested with Cy3-labeled complementary DNA to 
confirm the presence and the quality of the DNA barcode as shown in (c).  These DNA barcodes 
may be stored (under N2) for at least 6 months without degradation.  
 
 
 
 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
 
 
 
Supplementary Figure 4. Procedure for LF-IBBC preparation and operation. (a) Preparation of 
the microchip: 10 µl DNA-antibody conjugates and 10 µl wash buffer (3% BSA in PBS) were 
added to their respective chambers as illustrated. Subsequently, the conjugates are assembled 
onto the DNA array via DNA hybridization. (b) Operation of a LF-IBBC for blood sample 
analysis: wash buffer, Strep-Cy5 (Streptavidin-Cy5), Det Abs (Detection antibodies) and blood 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
are added to their respective chambers, and filter paper is used to initiate flow. An ELISA 
sandwich structure is generated where the fluorescence intensity of each stripe indicates the 
amount of the corresponding protein concentration in blood.  
 
 
Supplementary Figure 5. Cross-reactivity of the multiplex protein assay. Only one recombinant 
protein at 10 ng/ml was present in each sample to be detected (row). The red stripes indicate 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
proteins detected.  The green stripes are the references to spatially locate the red stripes and 
hence find the corresponding protein in Supplementary Table 2. 
 
 
 
 
Supplementary Figure 6. (a-c) Quantified results of 3 blood samples from different donors 
analyzed by LF-IBBC 40 min assays. 8 repeated barcodes were averaged and the standard 
deviations were generated to present error bars. (d) The control values are obtained by 
performing the same procedure as (a-c) except that the DNA-encoded capture antibodies were 
not patterned in the microchip, and so only background binding to the DNA oligomers was 
observed. Note that the y-axis of this plot is significantly expanded over that used for the blood 
samples.   
 
 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
 
 
 
 
 
 
 
 
Supplementary Tables 
 
Table 1. List of oligonucleotide sequences and terminal functionalization 
Name  Sequence       Tm 
A  5'- AAA AAA AAA AAA AAT CCT GGA GCT AAG TCC GTA-3'    57.9 
A'  5' NH3- AAA AAA AAA ATA CGG ACT TAG CTC CAG GAT-3'    57.2 
B 5'-AAA AAA AAA AAA AGC CTC ATT GAA TCA TGC CTA -3'    57.4 
B'  5' NH3AAA AAA AAA ATA GGC ATG ATT CAA TGA GGC -3'    55.9 
C 5'- AAA AAA AAA AAA AGC ACT CGT CTA CTA TCG CTA -3'   57.6 
C'  5' NH3-AAA AAA AAA ATA GCG ATA GTA GAC GAG TGC -3'    56.2 
D  5'-AAA AAA AAA AAA AAT GGT CGA GAT GTC AGA GTA -3'   56.5 
D'  5' NH3-AAA AAA AAA ATA CTC TGA CAT CTC GAC CAT -3'    55.7 
E  5'-AAA AAA AAA AAA AAT GTG AAG TGG CAG TAT CTA -3'    55.7 
E'  5' NH3-AAA AAA AAA ATA GAT ACT GCC ACT TCA CAT -3'    54.7 
F 5'-AAA AAA AAA AAA AAT CAG GTA AGG TTC ACG GTA -3'    56.9 
F'  5' NH3-AAA AAA AAA ATA CCG TGA ACC TTA CCT GAT -3'   56.1 
G 5'-AAA AAA AAA AGA GTA GCC TTC CCG AGC ATT-3'   59.3 
G'  5' NH3-AAA AAA AAA AAA TGC TCG GGA AGG CTA CTC-3'    58.6 
H  5'-AAA AAA AAA AAT TGA CCA AAC TGC GGT GCG-3'    59.9 
H'  5' NH3-AAA AAA AAA ACG CAC CGC AGT TTG GTC AAT-3'    60.8 
I  5'-AAA AAA AAA ATG CCC TAT TGT TGC GTC GGA-3'   60.1  
I' 5' NH3-AAA AAA AAA ATC CGA CGC AAC AAT AGG GCA-3'    60.1 
J 5'-AAA AAA AAA ATC TTC TAG TTG TCG AGC AGG-3'     56.5 
J'  5' NH3-AAA AAA AAA ACC TGC TCG ACA ACT AGA AGA-3'   57.5 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
K  5'-AAA AAA AAA ATA ATC TAA TTC TGG TCG CGG-3'     55.4 
K'  5' NH3-AAA AAA AAA ACC GCG ACC AGA ATT AGA TTA-3'    56.3 
L 5'-AAA AAA AAA AGT GAT TAA GTC TGC TTC GGC-3'   57.2 
L' 5' NH3-AAA AAA AAA AGC CGA AGC AGA CTT AAT CAC-3'   57.2 
M  5'-Cy3-AAA AAA AAA AGT CGA GGA TTC TGA ACC TGT-3'    57.6 
M'  5' NH3-AAA AAA AAA AAC AGG TTC AGA ATC CTC GAC-3'    56.9 
All sequences were purchased from Integrated DNA Technology (IDT) and purified through high performance 
liquid chromatography (HPLC). 
 
 
 
 
 
Table 2. Summary of antibodies and vendors 
DNA 
label 
Barcode 
sequence Name Primary antibody  Detection antibody Recombinant 
A'  1 CRP Mouse IgG2B Mab* (R&D***) Mouse IgG2B Mab (R&D) Sigma 
B'  2 MMP3 Goat IgG Pab** (R&D)  Goat IgG Pab (R&D) R&D 
C'  3 Serpin Mouse IgG1 Mab (R&D) Goat IgG Pab (R&D) R&D 
D'  4 G-CSF Mouse IgG1 Mab (R&D) Goat IgG Pab (R&D) R&D 
E'  5 MIF Mouse IgG1 Mab (R&D) Goat IgG Pab (R&D) ProSpec 
F'  6 EGF Mouse IgG1 Mab (R&D) Goat IgG Pab (R&D) ProSpec 
G'  7 CCL5 Mouse IgG1 Mab (R&D) Goat IgG Pab (R&D) R&D 
H'  8 VEGF Mouse IgG2B Mab (R&D) Goat IgG Pab (R&D) R&D 
I'  9 IL 8 Mouse IgG1 Mab (Biolegend) Mouse IgG1 Mab (Biolegend) Biolegend 
J'  10 IL-1 β Mouse IgG2b Mab (Biolegend) Mouse IgG1 Mab (Biolegend) Biolegend 
K'  11 IP10 Mouse IgG1 Mab (R&D) Goat IgG Pab (R&D) R&D 
L'  12 Control Mouse IgG1 Isotype control (R&D) N.A.  
*     Mab: monoclonal antibody 
**   Pab: polyclonal antibody 
*** R&D: R&D Systems 
 
 
 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
 
 
 
 
 
 
 
 
 
 
 
Supplementary Videos 
Supplementary video 1. Inertial focusing of blood cells when flowing at 0.25 µl/min powered 
by syringe pump. The width of narrow channel is 12 µm.  
Supplementary video 2. Inertial focusing of blood cells when flowing at 0.5 µl/min powered by 
syringe pump. The width of narrow channel is 12 µm.  
Supplementary video 3. Inertial focusing of blood cells when flowing at 1 µl/min powered by 
syringe pump. The width of narrow channel is 12 µm.  
Supplementary video 4. Inertial focusing of blood cells when flowing at 1.5 µl/min powered by 
syringe pump. The width of narrow channel is 12 µm.  
Supplementary video 5. On-chip plasma and cells separation powered by a filter paper. The 
width of narrow channel is 12 µm.  
Supplementary video 6. Self-replacement of dyes imaged at the end of narrow channel. 3 dyes 
were loaded to the LF-IBBC device. The video was taken at a speed of 0.5 frame/ min. 
 
Supplementary Methods 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
1. Microchip design and fabrication 
The microchip design shown in Supplementary Fig. 1 fully integrates filtration, mixing, 
separation, detection and absorption in one chip. 4 chambers were designed to load at most 4 
reagents for a complete ELISA assay as described in the text. They are connected by arrays of 
posts that increase flow resistance as well as filter impurities. The last post array is more 
compact to keep impurities originating in the chambers from clogging the narrow channel. 
 A serpentine channel following the post filter is designed to reduce possible 
concentration gradients within the assay channel cross section1.  It is not designed to mix the 
reagents from the separate microchambers.  Dean vortices are generated in the curvilinear 
channel to induce transverse flow, while only simple Poiseuille   flow occurs in straight channels 
of the microchip, pointing outwards in the same direction of the channel centerline. Without the 
serpentine channel, the fluorescence intensity across the width of a barcode stripe was not always 
uniform.  The following narrow channel with high aspect ratio is designed to separate plasma 
from blood cells through hydrodynamic focusing without sheath flow. The subsequent barcode 
readout channel incorporates the DNA-encoded antibody array to detect proteins of interest in 
situ by sandwich ELISA. At the end of the channel an array of posts with similar design is 
intended to increase resistance and facilitate connection to a filter paper through capillary flow. 
In the initial test, we used PDMS instead of NOA 63 to fabricate microfluidic devices. 
PDMS is not a good final solution for the microchip, because O2-plasma treated PDMS is 
difficult to remove from the glass slide, and such removal is necessary for the final fluorescence 
scanning step.  The pre-cure PDMS mixture of part A and part B (10:1; RTV 615, General 
Electric) was poured to a master and incubated at 80 °C for 40 min. The cured PDMS was cut off 
from the master and punched for access holes. Afterward the PDMS replica was cleaned by 
rinsing with isopropyl and deionized (DI) water followed with blow dry. After subsequent 
treatment with oxygen plasma at 25 W for 100 s to render the surface hydrophilic, the PDMS 
replica was bonded immediately to a DNA patterned array slide where the pattern was oriented 
perpendicularly to the microchannel. Because the barcodes cover the entire glass slide surface, 
no alignment is required during the processes of fabrication.  The fabrication process of NOA 63 
can be found in the Materials and Methods. This material is clear, colorless, has a low 
fluorescence background, thermally-stable and intrinsically sticky to a glass slide. After UV 
exposure of NOA 63, the surface is rendered significantly more hydrophilic with stability for 
more than 1 month and with a contact angle of 40o 2-3.  This is sufficient to allow solutions to 
spontaneously fill microchannels via capillarity. 
 
2. Cell focusing by inertial force 
Wall-repulsive forces have not been recognized as important for particle alignment in 
small-scale microfluidic channels. When the channel size is reduced to a certain dimension, wall-
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
Supplementary Figure 7. Schematic 
illustration of inertial forces in the cross 
section of narrow channel. The particle at 
channel center has an equilibrium of lift 
forces.
repulsive forces become prominent. In other publications, particles or cells are divided into at 
least two streams in the simplest straight channel by inertial forces, although complicated 
geometry designs have been introduced to focus particles into a single stream4-5. We incorporate 
the straight channel to separate plasma and cells not only because of the simplicity of design, and 
the resultant high separation efficiency but because it is robust and insensitive against 
perturbations to the flow conditions.  
When a cell or particle is flowing through the narrow channel, it experiences wall-
repulsive forces in a direction normal to the wall, and towards the microchannel center, due to 
stagnant flow close to the walls, as well as Saffman’s lift force toward channel walls due to shear 
stress (Supplementary Fig. 7). The interaction of those forces determines the transverse 
migration of the particle in the microchannel cross-section. Presumably under our current 
channel design and flow condition, the magnitude of the wall-repulsive forces dominates all 
other forces (including the Magnus force due to free rotation), resulting in particles migrating to 
the microchannel center and focusing into a single stream when exiting the narrow channel.  
 As shown in Supplementary Fig.2, single stream focusing only occurs when the width of 
narrow channel is less than ~12 µm. When the width is expanded to 16 µm, the cell stream tends 
to be divided into two. We previously observed 
that spherical cells can be well focused in a single 
stream. In consideration of erythrocytes being 
concave-like and more deformable, this focusing 
is robust and likely more affected by the channel 
geometry. For further reproduction of inertial 
focusing on other cell types, the width of the 
channel should not exceed twice the cell diameter. 
Meanwhile the cross-section should have an 
aspect ratio > 1.5. 
 
3. Patterning barcodes 
 We improved the quality of ssDNA 
barcode array in terms of uniformity and density 
of ssDNA per unit area. ssDNA oligomers 
purchased from IDT were dissolved in a solution containing DI water and DMSO (2:1 v/v). High 
concentrations of ssDNA (here, 266 µM) yielded very dense DNA on each bar.  
 A different PDMS/glass slide hybrid 
device was fabricated first for flow patterning 
ssDNA. As described before, the PDMS replica 
was created according to standard soft 
lithography, followed by bonding of PDMS to a 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
polylysine glass slide with thermal treatment at 80 °C for 1 hr. Each channel of the microfluidic 
device was filled with a unique ssDNA sequence. The ssDNA filled microchip was then placed 
in a desiccator to allow solvent (water and DMSO) to evaporate from the channel. The entire 
microchip with deposited ssDNA left in the channels was incubated at 80 °C for 4 hours. This 
thermal process crosslinks ssDNA to the surface of polylysine glass slide. The slide was 
separated from the PDMS and subsequently cleaned by dipping with DI water to remove un-
bonded ssDNA and other solids. Finally the slide was dried by N2 blowing and preserved in a 
desiccator. 
The compact antibody array was constructed by the DNA-encoded antibody library 
(DEAL) technique.  For this method, DNA-conjugated antibodies are assembled onto the ssDNA 
array through complementary DNA hybridization (Supplementary Fig. 3 and Supplementary Fig. 
4a)6. The ssDNA oligomers from A/A’ to M/M’ are optimized to be orthogonal to each other, 
ensuring one species of DNA linked antibody only localizes to a specific stripe with minimal 
cross-reactivity (Supplementary Table 1). Optimally, 3 strands of ssDNA linked to one antibody 
yields high affinity of the conjugate to pre-patterned complementary DNA without 
compromising antibody activity6. Sequence M is functionalized with M’-Cy3 as a reference, and 
provides registration for the remaining Cy5-labelled barcodes.  
4. Operation of the LF-IBBC chip 
 The LF-IBBC is initially prepared by converting the DNA array into an antibody array 
through the introduction of the cocktail of DNA-encoded antibodies (Supplementary Fig. 4a). A 
slice of filter paper with the appropriate size was inserted into the slot in contact with the channel 
end. We chose the thinnest filter paper (grade 50, Whatman) with thickness of 115 µm because 
the hardened surface and the strong wettability are desirable as a component in our chip. The 
filter paper with 1.5 cm2 can sustain wetting, and thus capillary-driven flow, for 2 hr. When we 
loaded wash buffer and DNA-antibody conjugates into the chambers, solutions filled all channels 
due to capillary force, within 5 min. Afterward the flow was supported capillarity from the filter 
paper. Once all solutions in the microchip were depleted, we stored the microchip in a 4 °C 
refrigerator.  
To perform a one-step assay of whole blood, the microchip with the DNA-encoded 
antibody array provides the starting point. We first loaded 7 µl whole blood sample, 5 µl 
detection antibodies, 5 µl streptavidin-cy5 and 15 µl 3% BSA wt/v in PBS to chamber 4, 3,2 and 
1, respectively (Supplementary Fig. 4b). The filter paper is then moved into the  channel end, 
which immediately allowed fluidic flow to occur. For these concentrations of reagents and blood, 
a 40 min assay time is sufficient to completely drain all four chambers.  
  
References 
Supplementary Material (ESI) for Lab on a Chip 
This journal is © The Royal Society of Chemistry 2010 
 
1. A. P. Sudarsan and V. M. Ugaz, Proc. Natl. Acad. Sci. U S A, 2006, 103, 7228-7233. 
2. S. H. Kim, Y. Yang, M. Kim, S. W. Nam, K. M. Lee, N. Y. Lee, Y. S. Kim and S. Park, 
Adv. Funct. Mater., 2007, 17, 3493-3498. 
3. E. P. Dupont, R. Luisier and M. A. M. Gijs, Microelectron. Eng., 2010, 87, 1253-1255. 
4. D. Di Carlo, D. Irimia, R. G. Tompkins and M. Toner, Proc. Natl. Acad. Sci. U S A, 2007, 
104, 18892-18897. 
5. J. F. Edd, D. Di Carlo, K. J. Humphry, S. Koster, D. Irimia, D. A. Weitz and M. Toner, 
Lab Chip, 2008, 8, 1262-1264. 
6. R. C. Bailey, G. A. Kwong, C. G. Radu, O. N. Witte and J. R. Heath, J. Am. Chem. Soc., 
2007, 129, 1959-1967. 
 
 


A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood

https://authors.library.caltech.edu/20396/2/C0LC00132E.PDF

A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main