Immuno-PCR on chip

The elucidation of the basic principles at molecular basis of many diseases demands analytical techniques that enable studies of biomolecular interactions, preferably at the level of single cells, or even single molecules. Methods that combine high-sensitivity detection with fast, robust and cost-efficient protocols for routine diagnostics and basic research applications are needed. The polymerase chain reaction (PCR) is a prime example of such an analytical tool because its almost exponential amplification efficiency enables the detection and quantification of small numbers of molecules of nucleic acids, using standard laboratory equipment.

However, most natural processes involve proteins and other non-nucleic acids, so the analysis of nucleic acids is not sufficient to explore biological principles. To extend the scope of PCR to the high-sensitivity detection of proteins, Sano et al. established the immuno-PCR (IPCR) method [2]. Taking advantage of specific conjugates comprising an antibody and a DNA marker fragment, IPCR combines the versatility of enzyme-linked immunosorbent assays (ELISAs) with the amplification power and sensitivity of the PCR. As a consequence, the limit of detection (LOD) of a given ELISA is, in general, enhanced 100–10 000 fold by the use of PCR as a signal amplification system (Figure 6). IPCR has been refined from a research method to a well established technique for routine applications in both fundamental and applied immunological research. Applications of IPCR in various field of biomedical research have been reported.

The general IqPCR protocol can be divided into two stages—target protein separation (stage 1) and barcode DNA detection (stage 2). In stage 1, antibodies against target protein coated to magnetic microparticles (MMP) is introduced into a microfluidic channel reactor. The sample fluid is then flowed into the channel along with functionalized gold nanoparticle probes linked to DNA universal primers (NP). Complex MMPtarget/protein-NP hybridise in a sandwiches only when target proteins are present in the sample fluid. The MMPs and MMP-target/protein-NP conjugates are then immobilized to the channel wall with a magnet while the supernatant is washed away with several column volumes of buffer. Subsequently, DNA strands are used as primers to be detected by PCR.

Immuno-PCR on chip

The system utilizes two types of particles: (i) magnetic microparticles coated with monoclonal antibodies and (ii) dual-functionalized gold nanoparticles surfaced by polyclonal antibodies and DNA marker fragment. Initial capture of a target analyte is performed by capture monoclonal antibodies coating on magnetic microparticles surface. Gold nanoparticles surfaced by detection polyclonal antibodies and 3’ alkylthiol-capped oligonucleotide accomplish subsequent detection. Oligonucleotide acts as DNA marker and can be amplified by Real-Time PCR with appropriate primers. Nucleotides, specific primers and probe, and a DNA polymerase are added and the marker is amplified by PCR for fluorescence signal generation. Successful application of IPCR relies upon the availability of polyclonal and monoclonal antibodies for a given antigen. On the other hand, as the DNA sequence can be chosen freely, it enables multiplex detection of various different antigens. The amount of DNA marker is then quantified using a fluorescence technique, Real-Time PCR. The number of PCR amplicons produced is proportional to the initial quantity of antigen to be detected. The technique here described is far less sensitive to error sources than is quantitative PCR (qPCR) due to there is just a single type of nucleic acid present in the sample to be amplified, which avoids mispriming or competition with other sequences. A standard curve has to be assayed with every qPCR assay, in order to minimize the background signal coming from matrix effects.

The IqPCR detection protocol is especially amenable for microfluidic implementation. The high sensitivity of the BCA method, stemming from signal amplification via DNA, allows low concentration levels to be detected while using small sample volumes. The high selectivity against background provides an efficient means of biofiltration and reduces sample pre-treatment requirements. Recently, it has been demonstrated this reaction on a silicon chip. This concept in combination with a LabOnFoil fabrication technology plus achieved concentration-PCR results will provide a never seen sensitivity, price and easy of performance for cancer disease monitoring.

The right balance between relevant, feasible and efficient research.
IKERLAN-IK4, Technological Research Centre. | J.M. Arizmendiarrieta N2 | 20500 Arrasate-Mondragon | Spain | jmruano@ikerlan.es