Microfluidic  immunoassays  as  rapid  saliva-­‐based  clinical  diagnostics  A  Review  on  Immunoassays  Regine  Labog  

ABSTRACT  

Point-­‐of-­‐care  diagnostics  have  benefited  immensely  from  microfluidic  devices.  Before  the  development  of  microfluidic  immunoassays  for  quantitatively  measuring  disease  through  biomarkers,  common  clinical  diagnostics  were  limited  to  binary  results  for  home  pregnancy  tests,  tuberculosis,  and  influenza.  This  paper  describes  an  advance  in  diagnostics  to  measure  a  biomarker  for  periodontal  disease  in  human  saliva.  This  research  could  be  developed  for  rapid,  reliable  measurement  of  analyzing  disease  markers  in  biological  fluids.  

Introduction   Peridontal disease affects one or more of the periodontal tissues: alveolar bone,

periodontal ligament, cementum, and gingiva. Unlike other diseases, periodontal disease

is a combination of multiple disease processes that share a common clinical

manifestation. If not treated, it leads to tissue deterioration, loss of connective tissue

attachment, and aleveolar bone loss. Furthering diagnostics research with microdevices

can eventually be used to frequently monitor episodic disease progression, enable early

diagnosis of a disease, or continuously assess therapeutic efficacy.

This paper uses microdevices to find matrix metalloproteinase-8 (MMP-8)1, a

major tissue-destructive enzyme in periodontal disease, in samples of saliva. To improve

the assay’s sensitivity to the enzyme, saliva pretreatment of mixing, incubation, and

enrichment, was included before placing the solution in the quantitative immunoassay.

The microchip electrophoretic immunoassay (µCEI) core of the device is based on

photolithographically fabricated molecular sieving gels to enrich the saliva sample and

later resolve a fluorescent antibody from the MMP-8 antigen-to-antibody complex.

Using microfluidics for point of care applications require a platform that is easy to

use, portable, user-friendly, and cheap. Colorimetric detection can fulfill these

requirments.2

Immunoassays  –  Advantages   Most biological procedures normally require solutions to be in an immobilized,

biochemically active phase.3 Immobilization is key, especially for heterogeneous

immunoassays because it affects specificity and sensitivity. Switching from the

macroscale to microscale depends on three main categories for biomolecular

immobilization: surface modification of microfluidic channel walls, packing microfluidic

channels with biomolecule-bearing beads, and packing microfluidic channels with

biomolecule-bearing porous slabs. For mircofluidic bioanalytical assays that do not use

an immobilized phase, an assay based on the rate of diffusion of antibody-antigen

complexes4 in solution as well as a technique for maintaining beads in place in a

recirculating flowstream without permanently immobilizing them is needed5.

Research on portable microfluidic devices for clinical diagnostics is a growing

industry because of its massive potential. These diagnostic devices would have lower

manufacturing costs, decreased sample size (here, a small amount of saliva is more than

enough), reproducible, and greater throughput. With the development of point-of-care

microfluidic diagnostics, clients could perform more complex diagnoses in their own

homes.

Immunoassays  –  Disadvantages   A significant disadvantage for microfluidic immobilization systems is its inherent

irreversibility. A channel surface that has been chemically modified is difficult to

remove, renew, or add an immobilized flexibility. This trait limits the flexibility of device

manufacturing since each device must be made with a specific immobilized biochemistry

for a specific application. These devices also take longer to construct as they are more

complex and the physics for macroscale machines differ from microscale devices due to

the laminar flow present in a microdevice.

Peridontal  Disease  Peridontal disease is a

progression of gingivitis and

its main cause is poor oral

hygiene. It destroys the

gingival fibers which are the

gum tissues that separate the

tooth from the peridontal

pocket6. Microorganisms

colonize these pockets and

further inflammate the gum

tissues and bone loss. If it is

not diagnosed and treated in

time, the microbic plaque

calcifies to form tartar and

must be removed above and

below the gum.

The prevalent method for measuring periodontal disease is with a periodontal

probe. It is placed between the gums and the teeth and slipped about 2 to 3mm below the

gum line. A subject with a peridontal pocket deeper than 7mm risks eventual tooth loss

over the years. However, this disease could go on without recognition for many years.

Types  of  Immunoassays   Microarrays are commonly used to perform immunoassays. An immunoassay

typically immobilizes antibodies and exposes them to a biological sample. It is separated

into four different types: direct-binding, sandwich (ELISA), competitive, and

displacement.

Direct-binding is when the antibody is labeled, normally fluorescently, and binds

with the target antigen. This method is not only quicker, but also avoids cross-

contamination with a secondary antibody. However, direct-binding requires using every

antibody which can be expensive and time-consuming. Also, some antibodies may not

qualify for direct-binding.

Sandwich (ELISA) quantifies the amount of antigen between the primary and

secondary antibodies. The target antigen must have at least two sites to bind to the

primary and secondary antibody since both must act in the sandwich. This restricts

sandwich assays to antigens with multiple binding sites for antibodies, such as proteins or

polysaccharides. However, sandwich is useful when there are low concentrations of

target antigens or high concentrations of contaminating proteins.

Competitive is used when a target antigen does not have any "matched pair"

antibodies to bind to. Here, the higher the antigen concentration, the weaker the signal

since fewer antibodies will be able to bind to the antigen in the well. The major

advantage is that it can use crude or impure samples to selectively bind any antigen

present. For the purposes of this paper, a competitive immunoassay was used due to the

amount of contaminants in saliva.

Displacement uses a micro capillary passage that immobilizes the antibodies to

the antigen of interest. As more antigen displaces the labeled antigen, the displaced

labeled antigen is detected.

Microfluidic  Electrophoresis  Capillary Electrophoresis (CE)7 uses a homogeneous phase immunoreaction,

which is normally very rapid due to mass transfer kinetics, followed by separation to

isolate and analyze the MMP-8 antigen. The unique fluid delivery capabilities of

microchip electrophoresis are necessary for automating immunoassays for use at the

point-of-care in the clinical environment. CE separates ionic species by their charge,

frictional forces, and hydrodynamic radius. Without CE, we would be unable to separate

the MMP-8 component from the rest of the saliva mixture.

The  Microchip  Electrophoretic  Immunoassay  (µCEI)   To include sample preparation and electrophoretic immunoassay on the same chip,

polymeric elements with certain physical patterns were photopatterned on class

microfluidic devices. The µCEI device consists of channels geared for specified

functions:

I. Sample Loading

II. Sample Enrichment

III. Rapid diffusive mixing of saliva with fluorescently labeled monoclonal antibody

[mAB] (MMP-8*)

IV. Subsequent Rapid Native Gel electrophoretic separation of MMP-8* from MMP-

8 complex.

 

Figure  1:  Multistep  Photopolymerization  of  µCEI  Device

Fabrication  of  the  µCEI    The three main regions fabricated were the size-exclusion membrane, a small pore-size

separation gel, and a larger pore-size loading gel.

Size-­‐Exclusion  Membrane  This portion was fabricated using laser photopolymerization of a solution of acrylamide

monomer, cross-linker, and photoinitiator using pressure-driven flow.

Pore-­‐Size  Separation  Gel  To define and localize the separation gel in the separation channel, all channels were

rinsed with a buffer and then pressure-loaded with the separation gel precursor solution.

UV photomasking was used to fabricate an intermediate porosity gel plug at the end of

the separation channel. Creating the plug resulted in a separation channel with separation

gel precursor and the elimination of bulk flow in the separation channel.

Pore-­‐Size  Loading  Gel  The loading gel was made using photopolymerization of an unmasked chip with a 100-W

UV lamp.

Layout  of  µCEI  Chip  The µCEI device is labeled for

sample (S), buffer (B), sample waste

(SW), buffer waste (BW), and the

fluorescently labeled monoclonal

antibody to MMP-8 (mAB*). After

a buffer priming step, the mAB* is

loaded into the size-exclusion

membrane followed by the saliva

sample, both through the large pore-

size loading gel. Once the two

solutions are mixed, an electric

potential is applied across the

membrane so that enriched species

go into the separation channel and

start the electrophoretic

immunoassay. Later, the electric

potential is switched to take out the

membrane from the current path.

Figure  2:  Layout  of  µCEI Chip  

Quantifying  µCEI  Assays   The sensitivity and dynamic range of µCEI assays allow us to vary the duration of

sample enrichment at the membrane or the magnitude of electric potential applied when

performing the enrichment step. Quantifying MMP-8 is the first step to moving away

from the binary nature of Point-of-Care clinical diagnostics and will help in monitoring

the disease activity in real time.

Macroscale  Comparison  of  Healthy  and  Periodontally  Diseased  Individuals.  While competitive immunoassay was used on the µCEI device, a regular colorimetric

sandwich ELISA was used in the macroscale to find the amount of concentration of

MMP-8 in saliva from the subjects. The severity of periodontal disease was assessed

through clinical examination, bleeding upon probing, pocket depth, and radiographic

bone loss. The most notable differences between healthy and diseased patients were in

the mean pocket depth and clinical attachment loss. A device capable of reporting

dynamic periodontal disease activity can also improve treatment by more effectively

timing the MMP inhibitor therapy since MMP-8’s active phase is correlated with

collagen deterioration.

Future  Directions  Researchers are motivated to achieve the potential of microfluidic immunoassays in

clinical diagnostics in order to take advantage of its miniaturization, integration, and

automation. However to do so, they must integrate the fields of material characterization,

fabrication, liquid transportation, surface modification, immobilization, and detection and

optimize them. The following are points to consider for the future development of

microfluidic immunoassays.

Mass  Production  for  Wide  Use  Although  PDMS  is  the  go-­‐to  polymer  for  microfluidic  research,  replicating  the  

fabrication  process  takes  hours  of  time  that  would  limit  product  manufacturing.  In  

order  to  make  massive  amounts  of  periodontal  disease  device  detectors,  other  

techniques  for  should  be  produced  such  as  injection  molding  and  embossing.  

Multiplexed  Assays  Single  chip  multiplexed  assays  are  an  important  feature  of  microfluidic  

immunoassays.  There  have  been  recent  developments  for  a  suspension  array  for  a  

multiplexed  immunoassay  with  Silica  Colloidal  Crystal  Beads  (SCCBs)8,9  that  show  

different  reflective  spectra  as  colors.  Combining  microfluidic  devices  with  SCCBs  has  

potential  for  clinical  applications  and,  regardless,  the  multiplexed  assay  will  remain  

the  dominant  method  of  commercialization  for  microfluidic  immunoassays.  

Surface  Modification  and  Immobilization  A  key  concern  for  immunoassays  is  the  nonspecific  adsorption  or  binding  to  

molecules  instead  of  analytes,  which  affects  the  sensitivity  and  selectivity  of  the  

assay.  The  competitive  immunoassay  is  a  good  alternative  for  impure  samples  and  

the  advances  in  surface  chemistry  and  functional  modification  has  been  studied  

extensively  enough  to  provide  a  solid  foundation  in  microfluidic  assays.  However  

there  is  still  difficulty  in  surface  modification  and  immobilization  of  these  materials.  

Purification  and  Concentration  As  mentioned  above,  the  complexity  and  small  amounts  of  antigens  in  samples  

require  purification  and  concentration  procedures.  Microbeads  can  help  improve  

sensitivity  and  helps  in  the  purification  process.  Their  increased  surface  area  and  

ease  of  use  provide  a  promising  method  for  one-­‐step  purification  and  concentration  

in  a  microfluidic  immunoassay.10  

Detection  Compared  to  other  microcomponents,  detection  systems  for  immunoassays  are  

bulky  and  expensive.  Although  some  integrated  detection  systems11  have  been  

developed,  the  cost,  sensitivity,  and  fabrication  processes  restrict  their  practical  

applications.  Thus,  developing  miniature,  portable,  and  inexpensive  detection  

systems  with  an  acceptable  sensitivity  for  microfluidic  devices  are  in  great  demand.  

Integration,  Packaging,  and  Price  Ultimately,  the  ideal  microfluidic  point  of  care  device  is  one  that  is  integrated,  

dispable,  and  cheap.  Most  devices  released  are  used  by  trained  lab  personnel  and  

other  auxiliary  machines  are  needed.  These  are  large  barriers  for  commercial  

applications  but  an  integrated  low-­‐cost  microfluidic  immunoassays  with  multiplex  

detection  function  is  possible,  with  further  research,  in  the  near  future.  

 

 

 

 

 

 

References  1. Herr, Amy, and Anson Hatch. "Microfluidic Immunoassays as Rapid Saliva-

based Clinical Diagnostics." Proceedings of the National Academy of Sciences 104.13 (2007): 5268-273. Print.

2. Taton, T. A., and C. Mirkin. "DNA Array De- Tection with Nanoparticle Probes."

Science 289 (2000): 1757-760. Print.

3. Noah, Malmstadt, and Hoffmann Alan. "“Smart” Mobile Affinity Matrix for Microfluidic Immunoassays." Lab Chip 4 (2004): 412-15. Print.

4. Hatch, A., and A. Kamholz. "Diffusion Immunoassay in Polyacrylamide Gels."

National Biotechnology 19 (2000): 461-65. Print.

5. Lettieri, G. "Separation Methods in Microanalytical Systems." Lab Chip 3 (2003): 34-39. Print.

6. D'Aiuto, F., M. Parkar, G. Andreou, J. Suvan, P.M. Brett, D. Ready, and M.S.

Tonetti. "Periodontitis and Systemic Inflammation: Control of the Local Infection Is Associated with a Reduction in Serum Inflammatory Markers." Journal of Dental Research 83.2 (2004): 156-60. Print.

7. Chiem, Nghia, and Jed Harrison. "Microchip Systems for Immunoassay: an Integrated Immunoreactor with Electrophoretic Separation for Serum Theophylline Determination." Clinical Chemistry 44.3 (1998): 591-98. Print.

8. Zhao, Yuanjin, Xiangwei Zhao, Cheng Sun, Juan Li, Rong Zhu, and Zhongze Gu.

"Encoded Silica Colloidal Crystal Beads as Supports for Potential Multiplex Immunoassay." Analytical Chemistry 80.5 (2008): 1598-605. Print.

9. Sun, Cheng, Xiang-Wei Zhao, Yuan-Jin Zhao, Rong Zhu, and Zhong-Ze Gu. "Fabrication of Colloidal Crystal Beads by a Drop-Breaking Technique and Their Application as Bioassays." Small 4.5 (2008): 592-96. Print.

10. Matsunaga, T., Y. Maeda, T. Yoshino, H. Takeyama, M. Takahashi, H. Ginya, J. Aasahina, and H. Tajima. "Fully Automated Immunoassay for Detection of Prostate-specific Antigen Using Nano-magnetic Beads and Micro-polystyrene Bead Composites, ‘Beads on Beads’." Analytica Chimica Acta 597.2 (2007): 331-39. Print.

11. Hofmann, Oliver, Xuhua Wang, John C. DeMello, Donal D. C. Bradley, and

Andrew J. DeMello. "Towards Microalbuminuria Determination on a Disposable Diagnostic Microchip with Integrated Fluorescence Detection Based on Thin-film Organic Light Emitting Diodes." Lab on a Chip 5.8 (2005): 863. Print.

12. De La Rica, Roberto, Antonio Baldi, César Fernández-Sánchez, and Hiroshi Matsui. "Single-Cell Pathogen Detection with a Reverse-Phase Immunoassay on Impedimetric Transducers." Analytical Chemistry 81.18 (2009): 7732-736. Print.

13. Gao, Yali, Guoqing Hu, Frank Y. H. Lin, Philip M. Sherman, and Dongqing Li.

"An Electrokinetically-Controlled Immunoassay for Simultaneous Detection of Multiple Microbial Antigens." Biomedical Microdevices 7.4 (2005): 301-12. Print.

14. Han, Jin-Hee, and Jeong-Yeol Yoon. "Reusable, Polyethylene Glycol-structured Microfluidic Channel for Particle Immunoassays." Journal of Biological Engineering 3.1 (2009): 6. Print.

15. Heyries, K., C. Mandon, L. Ceriotti, J. Ponti, P. Colpo, L. Blum, and C.

Marquette. "“Macromolecules to PDMS Transfer” as a General Route for PDMS Biochips." Biosensors and Bioelectronics 24.5 (2009): 1146-152. Print.

16. Lindmo, T., O. Bormer, J. Ugelstad, and K. Nustad. "Immunometric Assay by Flow Cytometry Using Mixtures of Two Particle Types of Different Affinity." Journal of Immunological Methods 126.2 (1990): 183-89. Print.

17. Qiu, Jingmin, Yun Zhou, Hui Chen, and Jin-Ming Lin. "Immunomagnetic

Separation and Rapid Detection of Bacteria Using Bioluminescence and Microfluidics." Talanta 79.3 (2009): 787-95. Print.

18. Suárez, Guillaume, Young-Hyun Jin, Janko Auerswald, Stefan Berchtold, Helmut F. Knapp, Jean-Marc Diserens, Yves Leterrier, Jan-Anders E. Månson, and Guy Voirin. "Lab-on-a-chip for Multiplexed Biosensing of Residual Antibiotics in Milk." Lab on a Chip 9.11 (2009): 1625. Print.

19. Wang, H., S. Meng, K. Guo, Y. Liu, P. Yang, W. Zhong, and B. Liu.

"Microfluidic Immunosensor Based on Stable Antibody-patterned Surface in PMMA Microchip." Electrochemistry Communications 10.3 (2008): 447-50. Print.

20. Yager, Paul, Thayne Edwards, Elain Fu, Kristen Helton, Kjell Nelson, Milton R.

Tam, and Bernhard H. Weigl. "Microfluidic Diagnostic Technologies for Global Public Health." Nature 442.7101 (2006): 412-18. Print.

21. Yager, Paul, Thayne Edwards, Elain Fu, Kristen Helton, Kjell Nelson, Milton R.

Tam, and Bernhard H. Weigl. "Microfluidic Diagnostic Technologies for Global Public Health." Nature 442.7101 (2006): 412-18. Print.

                                                                                                               1 Microfluidic immunoassays as rapid saliva-based clinical diagnostics Amy E. Herr†‡, Anson V. Hatch†, Daniel J. Throckmorton†, Huu M. Tran†, James S. Brennan†, William V. Giannobile§, and Anup K. Singh† †Biosystems Research Department, Sandia National Laboratories, Livermore, CA 94550; and §Michigan Center for Oral Research, School of Dentistry, University of Michigan, Ann Arbor, MI 48106 Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved January 11, 2007 (received for review August 21, 2006)/5268–5273 ! PNAS ! March 27, 2007 ! vol. 104 ! no. 13 2 Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Scanometric DNA array de- tection with nanoparticle probes. Science 2000, 289(5485), 1757e1760. 3 “Smart” mobile affinity matrix for microfluidic immunoassays Noah Malmstadt, Allan S. Hoffman* and Patrick S. Stayton* Department of Bioengineering, University of Washington, Seattle, WA 98195, USA Received 27th November 2003, Accepted 12th March 2004 First published as an Advance Article on the web 6th April 2004 Lab Chip, 2004, 4, 412–415 4 A. Hatch, A. E. Kamholz, K. R. Hawkins, M. S. Munson, E. A. Schilling, B. H. Weigl and P. Yager, Nat. Biotechnol., 2001, 19, 461–465. 5 G. L. Lettieri, A. Dodge, G. Boer, N. F. de Rooij and E. Verpoorte, Lab Chip, 2003, 3, 34–39. 6 D'Aiuto F, Parkar M, Andreou G, Suvan J, Brett PM, Ready D, Tonetti MS. (2004). Periodontitis and systemic inflammation: control of the local infection is associated with a reduction in serum inflammatory markers. J Dent Res. 83(2):156-60.

7 Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination Nghia H. Chiem and D. Jed Harrison*, Clinical Chemistry 44:3 591–598 (1998) 8 Zhao, Y,; Zhao, X. W.; Sun, C.; Li, J.; Zhu, R.; Gu, Z. Z. Encoded silica colloidal crystal beads as supports for potential multiplex immunoassay. Anal. Chem. 2008, 80(5), 1598e1605. 9 Sun, C.; Zhao, X. W.; Zhao, Y. J.; Zhu, R.; Gu, Z. Z. Fabrication of colloidal crystal beads by a drop-breaking technique and their applica- tion as bioassays. Small 2008, 4(5), 592e596. 10 Matsunaga, T.; Maeda, Y.; Yoshino, T.; Takeyama, H.; Takahashi, M.; Ginya, H.; Aasahina, J.; Tajima, H. Fully automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micro-polystyrene bead composites, ‘Beads on Beads’. Anal. Chim. Acta 2007, 597(2), 331e339. 11 Hofmann, O.; Wang, X.; deMello, J. C.; Bradley, D. D. C.; deMello, A. J. Towards microalbuminuria determination on a disposable diagnostic microchip with integrated fluorescence detection based on thin-film or- ganic light emitting diodes. Lab Chip 2005, 5(8), 863e868.