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Biomarker detection via lab-on-a-chip integrated immunoaffinity approach for fluorescence and mass spectrometry readout

Författare:
Publiceringsår: 2011
Språk: Engelska
Sidor:
Dokumenttyp: Doktorsavhandling

Sammanfattning

Popular Abstract in English

The human plasma proteome containing disease-correlated protein biomarkers

reflects the pathological and physiological changes relating to diseases. These

biomarkers are of interest to aid in diagnosis, prognosis and monitoring of diseases

such as cancer. Because of the interests in the disease-correlated biomarkers, plasma,

although is a complex biological sample, spanning over 10 orders of magnitude in

protein concentrations, is the most widely used clinical specimen for analysis.

Detecting these biomarkers represents biological and technical challenges in clinical

diagnostics.

Lab-on-a-chip approach could provide insights for a simplified and improved

detection platform, eliminating the laborious sample preparations compulsory in

most conventional methods. With regards to protein biomarker detection for use in

point-of-care settings, recent advancements, paved by the microfluidic technology

have been demonstrated. Microfluidic devices, manipulating fluids in minute

volumes in the range of micro to nanoliter or less, offer insights into enabling a fully

integrated, high throughput, cost-effective, rapid ‘sample to answer’, miniaturized

immunoassay systems. Microfluidic whole-blood immunoassays hold potential in

point-of-care diagnostics application, which has brought us to the exploration of

combining the microfluidic technology and antibody microarrays described in this

thesis work. At the Department of Electrical Measurements, we have earlier

developed a sandwich antibody microarray on 3D structured porous silicon surfaces.

Using this method, in this thesis, the focus in the beginning was on detecting the

prostate cancer biomarker, Prostate Specific Antigen (PSA) in plasma samples. We

have also shown a signal enhancement step utilizing europium nanoparticles to

improve the detection limit of our microarray platform for future development of a

multiplex prostate cancer biomarker detection platform. Along the line, microfluidic

technology specifically utilizing ultrasonic standing wave forces present in the acoustic field has been extensively explored at the department. Since plasma is

highly demanded in clinical diagnostics, we designed an acoustophoresis-based

microchip capable of separating plasma from undiluted whole blood. We

demonstrated the potential application of the acoustic plasmapheresis microchip by

linking it to a subsequent off line detection of PSA on our porous silicon antibody

microarray. We then moved forward with the idea of combining on-chip plasma

separation and a microarray immunoassay, in which a manifold encompassing both

platforms in an integrated manner was designed. We showed the capability of rapid

immunoaffinity-capturing of PSA directly from whole blood samples, which hold

promise of establishing a ‘whole blood sample to answer’ assay.

Another interesting lab-on-a-chip application is in the field of mass spectrometrybased

plasma proteomics which focuses on reducing the laborious sample

preparation step prior to mass spectrometry analysis. Generally, the chip-based

capillary electrophoresis separation, on-chip protein digestion and chip for direct

infusion i.e. for ESI/MS interface are among the much earlier explored applications

in the field of proteomics. In clinical proteomics, where target proteins/ peptides are

often known, mass spectrometry analysis in combination with a pre step of

immunoaffinity separation, could eliminate the need for extensive separation

methods, offering a simplified quantitative analysis path for detection of protein

biomarkers. At the Department of Electrical Measurements, we use lab-on-a-chip

integrated immunoaffinity approach in combination with mass spectrometry that

could offer insights in specifically detecting protein/peptide biomarkers from

complex biological samples. We realized the need for integrated platforms that

include reduction in sample complexity, minimize sample transfers and the

importance to be able to enrich the targeted protein of interest prior to mass

spectrometry analysis. Using our microfabricated Integrated Selective Enrichment

Target which is compatible for use in any MALDI instrument, we developed

immunoaffinity, on-target protein biomarker digestion and sample cleanup protocols

to be able to show detection of a protein biomarker, PSA on an integrated platform.

This opens potential application for use in the development of SRM analysis for

quantitative measurements of proteins. For detecting peptide from complex samples,

we developed a porous silicon array-based immunoMALDI protocol demonstrated

by the detection of Angiotensin I peptide from plasma samples.
The blood/plasma protein biomarker profile represents the pathological and physiological changes relating

to human diseases. However, the plasma proteome reflects a high degree of complexity that results in a

challenge for most analytical methods in clinical diagnostics.

The focus in this thesis is the development and application of several integrated platforms to detect already

established protein/peptide biomarkers from blood and plasma (Prostate Specific Antigen, PSA and

Angiotensin I, Ang I). The integrated platforms that are described in the papers included in this thesis

utilize protein/peptide immunoaffinity-capturing since it offers insights into dealing with the challenges of

the plasma proteome analysis by reducing complexity and enriching the proteins/peptides of interest.

Several integrated platforms were developed with the overall main aim of detecting biomarkers from

complex biological samples:

• porous silicon antibody microarray with signal amplification step (Paper I)

• microfluidic whole blood immunoassay (Paper II and III)

• integrated protein immunoaffinity capturing with microfabricated proteomic sample processing

platform (ISET) enabling a direct interface to MALDI MS/MS analysis (Paper IV)

• integrated peptide immunoaffinity capturing via porous silicon array-based immuno-MALDI

(Paper V) enabling a direct interface to MALDI MS/MS analysis

Ultimately, these platforms target future use in point-of-care settings (Paper I-III) and immuno-MALDI

mass spectrometry immunoassays for identification and quantitative measurements of biomarkers using

isotope labelled standards (Paper V) as well as outlook for use in the development of SRM/MRM assays

(Paper IV).

Disputation

2011-12-16
10:00
E:1406, E-huset, Ole Römers väg 3, Lund University, Faculty of Engineering
  • Fred Regnier (Prof.)

Nyckelord

  • Medical Engineering
  • immunoaffinity
  • affinity prefractionation
  • immunoMALDI
  • solid-phase extraction
  • protein digestion
  • ISET
  • antibody microarray
  • signal amplification
  • fluorescence
  • microfluidics
  • lab on a chip
  • separation
  • acoustophoresis
  • ultrasound
  • acoustic radiation force
  • plasma
  • proteomics
  • MALDI mass spectrometry
  • immunoassay
  • porous silicon.

Övriga

Published
  • Thomas Laurell
  • ISSN: 0346-6221
  • ISBN: 978-91-7473-217-7

Box 117, 221 00 LUND
Telefon 046-222 00 00 (växel)
Telefax 046-222 47 20
lu [at] lu.se

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Organisationsnummer: 202100-3211
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