We report here the fabrication and validation of a novel 3D-printed, automated immunoarray to detect multiple proteins with ultralow detection limits. This low cost, miniature immunoarray employs electrochemiluminescent (ECL) detection measured with a CCD camera and employs touch-screen control of a micropump to facilitate automated use. The miniaturized array features prefilled reservoirs to deliver sample and reagents to a paper-thin pyrolytic graphite microwell detection chip to complete sandwich immunoassays. The detection chip achieves high sensitivity by using single-wall carbon nanotube-antibody conjugates in the microwells and employing massively labeled antibody-decorated RuBPY-silica nanoparticles to generate ECL. The total cost of an array is $0.65, and an eight-protein assay can be done in duplicate for $0.14 per protein with limits of detection (LOD) as low as 78-110 fg mL in diluted serum. The electronic control system costs $210 in components. Utility of the automated immunoarray was demonstrated by detecting an eight-protein prostate cancer biomarker panel in human serum samples in 25 min. The system is well suited to future clinical and point-of-care diagnostic testing and could be used in resource-limited environments.

Detecting cancer at an early stage of disease progression promises better treatment outcomes and longer lifespans for cancer survivors. Research has been directed towards the development of accessible and highly sensitive cancer diagnostic tools, many of which rely on protein biomarkers and biomarker panels which are overexpressed in body fluids and associated with different types of cancer. Protein biomarker detection for point-of-care (POC) use requires the development of sensitive, noninvasive liquid biopsy cancer diagnostics that overcome the limitations and low sensitivities associated with current dependence upon imaging and invasive biopsies. Among many endeavors to produce user-friendly, semi-automated, and sensitive protein biomarker sensors, 3D printing is rapidly becoming an important contemporary tool for achieving these goals. Supported by the widely available selection of affordable desktop 3D printers and diverse printing options, 3D printing is becoming a standard tool for developing low-cost immunosensors that can also be used to make final commercial products. In the last few years, 3D printing platforms have been used to produce complex sensor devices with high resolution, tailored towards researchers’ and clinicians’ needs and limited only by their imagination. Unlike traditional subtractive manufacturing, 3D printing, also known as additive manufacturing, has drastically reduced the time of sensor and sensor array development while offering excellent sensitivity at a fraction of the cost of conventional technologies such as photolithography. In this review, we offer a comprehensive description of 3D printing techniques commonly used to develop immunosensors, arrays, and microfluidic arrays. In addition, recent applications utilizing 3D printing in immunosensors integrated with different signal transduction strategies are described. These applications include electrochemical, chemiluminescent (CL), and electrochemiluminescent (ECL) 3D-printed immunosensors. Finally, we discuss current challenges and limitations associated with available 3D printing technology and future directions of this field.

Low cost, miniaturized assay platforms that work with small sample volumes, high sensitivity and rapid detection will have high value in future biomolecular diagnostics. Herein we report an automated, 3D printed electrochemiluminescent (ECL) immunoarray integrated with a nanostructured pyrolytic graphite sheet (PGS) microwell chip configured to detect 2 proteins simultaneously from complex liquid samples with high sensitivity and selectivity. Assays are done in 18 min at cost of < $1.00 using 1–2 microliters of sample. 3D printed microfluidic array design integrates reagent and sample chambers with rapid ECL detection. A commercial programmable syringe pump used with a preset program allows pump to pause and resume reagent delivery as required for completion of the sandwich immunoassays. Nanostructured surfaces feature antibody-decorated single wall carbon nanotube forests on PGS chip microwells, and sensitivity is amplified via massively labeled RuBPY-silica nanoparticles for detection. Prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) were measured simultaneously from human serum on the immunoarray with detection limits 150 fg mL−1 for PSA and 230 fg mL−1 for PSMA, with dynamic ranges up to 5 ng mL−1. Validation of the immunoarray by measuring these proteins in human serum showed good correlation with single protein ELISA. These 3D printed platforms can be easily adapted to multiple applications and configurable CAD files for the immunoarray can be downloaded from our lab’s website.