Wang et al. Microsystems & Nanoengineering (2021) 7:65 Microsystems & Nanoengineering https://doi.org/10.1038/s41378-021-00291-w www.nature.com/micronano ARTICLE Open Access Monolithic integration of nanorod arrays on microfluidic chips for fast and sensitive one-step immunoassays Ye Wang1, Jiongdong Zhao1,YuZhu2, Shurong Dong1,3,YangLiu1, Yijun Sun1, Liling Qian4, Wenting Yang5 and ✉ Zhen Cao 1,3 Abstract Here, we present integrated nanorod arrays on microfluidic chips for fast and sensitive flow-through immunoassays of physiologically relevant macromolecules. Dense arrays of Au nanorods are easily fabricated through one-step oblique angle deposition, which eliminates the requirement of advanced lithography methods. We report the utility of this plasmonic structure to improve the detection limit of the cardiac troponin I (cTnI) assay by over 6 × 105-fold, reaching down to 33.9 fg mL−1 (~1.4 fM), compared with an identical assay on glass substrates. Through monolithic integration with microfluidic elements, the device enables a flow-through assay for quantitative detection of cTnI in the serum with a detection sensitivity of 6.9 pg mL−1 (~0.3 pM) in <6 min, which was 4000 times lower than conventional glass devices. This ultrasensitive detection arises from the large surface area for antibody conjugation and metal-enhanced fluorescent signals through plasmonic nanostructures. Moreover, due to the parallel arrangement of flow paths, simultaneous detection of multiple cancer biomarkers, including prostate-specific antigen and carcinoembryonic antigen, has been fulfilled with increased signal-to-background ratios. Given the high performance of this assay, 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; together with its simple fabrication process that is compatible with standard mass manufacturing techniques, we expect that the prepared integrated nanorod device can bring on-site point-of-care diagnosis closer to reality. Keywords: Nanorods; Plasmonics; Immunoassay; Oblique angle deposition Introduction performed in 96-well plates, require complicated operating Immunoassays have been widely explored and have procedures, have long incubation times, and need bulky become the gold standard for detecting myriad biomarkers instruments, which are incompatible with the requirements in disease treatment and clinical diagnostics1,2.Itemploys of on-site diagnostics and point-of-care testing5. In recent specific bindings of antibodies and antigens for quantitative years, the advances in lab-on-a-chip technology have con- analysis of proteins in biological samples. Fluorescent tributed to the progress of miniaturized bioanalytical devi- – immunoassays are one of the most popular and well- ces6 8. Many portable, rapid, and cost-effective microfluidic developed analytical methods that measure fluorescence to chips have been developed to address special issues in quantify target antigens in clinical laboratories3,4.However, fluorescent assays, such as labor-intensive preparation conventional fluorescent immunoassays are routinely procedures, nonspecificbinding,ordifficulty in miniatur- ization. For instance, Gervais and Delamarche proposed a plastic microfluidic chip for a one-step immunoassay9.By Correspondence: Zhen Cao ([email protected]) integrating various fluidic components and reagents on 1College of Information Science and Electronic Engineering, Zhejiang University, chips, inflammation and the cardiac marker C-reactive ’ − 310027 Hangzhou, People sRepublicofChina protein (CRP) were detected at a concentration <1 ng mL 1 2Suzhou Institute of Nano-tech and Nano-Bionics, Chinese Academy of Sciences, 215123 Suzhou, People’s Republic of China in 5 min. Song et al. presented a multiplexed volumetric Full list of author information is available at the end of the article © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Wang et al. Microsystems & Nanoengineering (2021) 7:65 Page 2 of 10 fi bar-chart chip for the rapid quanti cation of protein bio- Nanorods markers with a simple operation and easy read-out10.They further utilized gas competition and reported a digital Sample inlet Reaction chamber volumetric bar-chart chip (DV-chip) for quantitative diag- nostics with ultralow detection limits (~0.1 pM) that are readable with the naked eye11.Othermicrofluidic concepts – for miniaturized bioassays include lab-on-paper12 14,lab- – on-a-disc15 17, lab-on-a-syringe18, and lab-on-a stick19. Despite substantial progress made toward the improve- ment of assay throughput, decreases in costs and time, and operational simplification, the sensitivity or detection limit remains one of the core parameters that can be tre- mendously increased using nanostructures or nanoparticles dAb deposition zone Capillary pump due to the metal-enhanced fluorescence (MEF) effect. With fl fl the rapid development of nanotechnology, various nanos- Fig. 1 Conceptual illustration of the ow-through micro uidic device integrated with Au nanorods for one-step immunoassay tructures are fabricated and dramatically improve the sen- of protein biomarkers. The serum sample containing target sitivity of immunoassays. Typical examples include biomarkers is introduced and conjugated with the fluorescent dAb plasmonic gold-on-gold (Au/Au) films20, nanoparticles21,22, prepatterned in the deposition zone. The complex flows through the – nanopillars23 25, nanorods26,27, and nanowells28.Among reaction chamber and is captured by cAb conjugated with dense fl those techniques, advanced nanofabrication techniques arrays of embedded Au nanorods. The enhanced uorescent signals are measured and analyzed through a microscope equipped with a such as e-beam lithography (EBL) or nanoimprinting are CCD camera. The flow rate and total reaction time are defined by the commonly employed to pattern the desired nanostructures capillary pump on the substrate. However, EBL is limited by expensive equipment and suffers from low throughput. Nanoim- printing may involve complicated preparation of the imprint template and raise issues such as mold deformation Au nanorods enable a flow-through immunoassay for cTnI during the process29. Moreover, colloidal lithography is within 6 min with a decrease in the detection limit from − − proposed using the self-assembly of colloidal particles as 27.0 ng mL 1 (~1.1 nM) down to 6.9 pg mL 1 (~0.29 pM). etching masks for nanopatterning30. Nevertheless, it A layout of multiplex flow paths is also designed for the requiresacumbersomeetchingprocessandmayfaceissues simultaneous detection of highly sensitive cancer bio- such as its uniformity over large areas. Bottom-up solution markers prostate-specific antigen (PSA) and carcinoem- synthesis is not suitable for mass production and suffers bryonic antigen (CEA). from complex operating procedures. Therefore, it is of The conceptual design of our fluidic chip integrated with utmost importance to find effective nanostructures with nanorods is shown in Fig. 1 for highly sensitive immu- easier fabrication schemes to improve the MEF and detec- noassays. The sample is introduced from the inlet and tion limit of assays. pumped through the flow resistor to the detection antibody Previously, we reported the easy fabrication of nanorod (dAb) zone, where the fluorescent dAb is predeposited on arrays through oblique angle deposition (OAD), and these the glass substrate. The target antigen in the sample binds nanostructures have been extensively studied and applied in with the dAb as it flows to the reaction chamber. In this many fields, such as surface-enhanced Raman spectro- chamber, the complex is bound to the capture antibody scopy31,32 and high-performance capillary electrophor- (cAb), which is covalently conjugated with dense arrays of esis33,34. Herein, we present a simple and sensitive embedded nanorods. The fluorescent signal is boosted due to microfluidic immunoassay chip that integrates Au nanorods MEF and detected by microscopy for further analysis. in microchannels for the quantitative detection of cardiac troponin I (cTnI). These nanostructures are selectively Results and discussion immobilized on the detection zone of the chip through one- Simulations step evaporation, which eliminates the requirement for MEF refers to the phenomenon that fluorescence emis- advanced lithography tools or complicated processes. The sions of fluorophores are significantly increased when they plasmonic substrates afford fluorescence enhancement and are in close proximity to conducting metallic particles, col- provide a large surface area for antibody conjugation. The loids, or surfaces. This is mainly due to the strong and close- sensitivity of the cTnI assay is greatly improved by over 6 × range interactions between these fluorescent molecules with − 105-fold, with a detection