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    • thip br Acknowledgements This work was partly supported

    2018-10-30


    Acknowledgements This work was partly supported by the Center of Innovation Program from Japan Science and Technology Agency, JST, and was also partly supported by the Research Institute for Science and Engineering, Waseda University (14P56).
    Introduction Alpha fetoprotein (AFP), a glycoprotein with a molecular weight of approximately 70kDa, is the most abundant plasma protein found in human fetus. However, the plasma level decreases rapidly to normal adult levels during the first year of life. Normally, AFP is present only at rather low levels in the blood of healthy people (reference value <10ng/ml) [1]. Certain types of cancers and liver diseases are known to lead to increased AFP levels. Accordingly, AFP is widely used as a serum biomarker for hepatocellular carcinoma (HCC) screening in patients [2] and for HCC diagnosis [3]. Unusual AFP levels during pregnancy in the maternal blood or in the amniotic fluid are taken as serological soft markers for congenital malformation (e.g. open neural tube defects, anencephaly) or chromosome anomalies. An unusually low value (depending on the stage of gestation) might serve as an indication of the presence of the Down-Syndrome (Trisomie-21) in the developing fetus [4–7]. As of today several techniques have been developed for AFP detection, for example electrochemical immunosensors [8,9], colorimetric detection [10–12] and piezoelectric biosensors [13]. Most of these techniques utilize covalent binding reactions to immobilize the antibody onto the sensing surface [8–11]. Immobilization via covalent binding requires several steps including surface preparation and activation to provide appropriate reactive surface groups, and antibody immobilization. In general, immobilization by practically all methods know today is a multi-step process which is time and resource consuming and increases the risk of quality problems in the production process. The surface area available for binding of the probe thip is an important factor for immobilization. When self assembled monolayers are employed, the surface density of capture molecules is intrinsically limited. Additionally, at high surface concentrations the intermolecular distances of the bound molecules are decreased, which might influence the accessibility for target molecules. A too dense binding can result in steric hindrance and might reduce the efficiency of immobilization process [14,15]. To increase the surface density of accessible biomolecules on a sensor surface, immobilization based on surface-attached very thin hydrogel pads has been developed which allow a more three dimensional arrangement of the molecules [15,16].
    Materials and methods
    Results and discussion
    Conclusion In this study, a rapid photo-immobilization process based on C,H insertion reactions was successfully applied to fabricate protein microarrays for the detection and quantification of alpha fetoprotein (AFP). Microarray printing of a mixture of probe molecules and photopolymer followed by brief UV-exposure is sufficient to generate protein microarrays, which allow quantification of AFP over the whole concentration range required in diagnostics, i.e. between 5 and 100ng/ml. The sensitivity of the developed chip is so high that all analyte solutions need to be diluted by 100 times in order to avoid overloading of the surface, which would prevent quantification. After dilution a linear response between concentration and read-out signal was observed.
    Acknowledgments The work was partially supported by the National Nanotechnology Center (NANOTEC) through the Center of Excellence Network program at Mahidol University. Partial support by the Thailand Center of Excellence for Life Sciences (TCELS) is gratefully acknowledged. A PhD scholarship for SY was supported by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0246/2551). In Germany, the project was partially supported by the BMBF (German Ministry of Education and Research) FKZ 0315999C. We would like to acknowledge Mr. Holger Frey for his kind assistance and his advice for the printing of the microarrays.