Ultrasensitive 3D Printed Immunoarrays for Protein Detection Down to Single Cell Levels

Accurate, sensitive measurements of proteins are crucial for modern biomedical research, with applications in basic research, medical diagnostics, therapy guidance, and drug development. Fast, selective, low cost detection of multiple proteins is important for public health and a key component of future personalized medicine. There are >50,000 gene product proteins (omitting post-translational modifications and IgGs) in human plasma, and large subsets in serum, urine, and saliva. However, techniques such as LC-MS/MS, immunoassays and protein counting can reliably detect <2000 of these proteins and are quite expensive. This paper addresses the challenge to develop low cost, automated 3D-printed immunoarrays for liquid protein samples and cells with LODs <0.1 fg/mL. We have developed prototype multiplexed microfluidic immunoarrays with sensitivity better than protein counting applicable to plasma, serum, saliva, urine, cell lysates and cell cultures. Protein detection at the single cell level was also achieved. Unique detection features including nanostructured sensing, array design, and protocol optimization provide large decreases in LODs and greatly enhanced sensitivities. Our prototypes are based on chemiluminescence (CL) and amperometric detection. Our CL array achieved LODs of 0.1 fg/mL for membrane protein DSG3, as well as VEGF-C and VEGF-D in conditioned media of cell cultures. This LOD is 0.8 zeptomolar, or ~100 molecules of DSG3 in 90 mL and results from 30 trillion capture antibodies (Ab1) in array detection cylinders using an ultrabright femto-luminol CL reagent activated by H2O2 and poly-(horseradish peroxidase) (poly-HRP, 400 HRPs/molecule). Femto-luminol combined with poly-HRP provides very high CL signal amplification and S/N using the 400 HPR polymer.Proteins are also efficiently captured in detection cylinders with 30 trillion antibodies, combined with the ultrabright CL reagent and rigorous protocol optimization to prove unprecedented LODs. Microprocessor automation can achieve fast multiplexed assays with these immunoarrays at <$0.50/protein. We are also exploring ultra-high sensitivity amperometric detection in these systems with similar sensitivity enhancement principles.