Biomarkers such as nucleic acids and proteins are powerful indicators of the physiological state of organisms, including humans. Thus, monitoring the presence rather than changes in the levels of biomarkers can help in the diagnosis of a disease or in the assessment of its progression. The higher the sensitivity and specificity of a given biomarker detection method, the earlier a diagnosis can be established. Commonly, the detection of biomarkers requires many steps, and each step contributes to the overall sensitivity of the method. In order to increase the sensitivity, improvements in all steps (from sample preparation to detection) are required. The aim of this dissertation was to study and develop innovative and highly sensitive methods for the detection of biomarkers.

Polymerase chain reaction (PCR) was the chosen technique for the final signal readout for all studies, due to its high sensitivity and reproducibility. While PCR has an extremely high sensitivity, it is limited by the DNA isolation method. In an improvement of the sample preparation approach, we have developed a novel method to recover small diagnostic DNA fragments from blood serum using compaction precipitation. Small, circulating diagnostic DNA fragments are useful in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. We were able to isolate and concentrate small DNA from serum, and increase the sensitivity of detection by more than four orders of magnitude.

While searching for further PCR optimization methods, we came across highly publicized reports in high-impact journals about enhancement of PCR by gold nanoparticles. However, we showed that gold nanoparticles do not enhance the specificity of PCR, but rather suppress the amplification of longer products while favoring amplification of shorter products, independent of specificity.

The current methods for protein detection are not as sensitive and specific as methods for DNA detection. We have developed a novel, extremely sensitive PCR based method of detecting protein and RNA using magnetic nanoparticles labeled with both DNA and antibody. This method has the ability to detect up to five different biomarkers simultaneously, and we can measure the presence of fewer than 100 immunomagnetic nanoparticles by real-time PCR.