The Use of Nanoarrays for Highly Sensitive and Selective Detection of Human Immunodeficiency Virus Type 1 in Plasma Ki-Bum Lee, † Eun-Young Kim, ‡ Chad A. Mirkin,* ,† and Steven M. Wolinsky* ,†,‡ Department of Chemistry and The Institute for Nanotechnology, Northwestern UniVersity, EVanston, Illinois, and DiVision of Infectious Diseases, The Feinberg School of Medicine, Northwestern UniVersity, Chicago, Illinois Received June 28, 2004; Revised Manuscript Received August 6, 2004 ABSTRACT Arrays of antibodies with well-defined feature size and spacing are necessary for developing highly sensitive and selective immunoassays to detect macromolecules in complex solutions. Here we report the application of nanometer-scale antibody array-based analysis to determine the presence of the human immunodeficiency virus type 1 (HIV-1) in blood samples. Dip-pen nanolithography (DPN) was used to generate nanoscale patterns of antibodies against the HIV-1 p24 antigen on a gold surface. Feature sizes were less than 100-nanometers, and the activity of the antibody was preserved. HIV-1 p24 antigen in plasma obtained directly from HIV-1-infected patients was hybridized to the antibody array in situ, and the bound protein was hybridized to a gold antibody-functionalized nanoparticle probe for signal enhancement. The nanoarray features in the three-component sandwich assay were confirmed by atomic force microscopy (AFM). Demonstration of measurable amounts of HIV-1 p24 antigen in plasma obtained from men with less than 50 copies of RNA per ml of plasma (corresponding to 0.025 pg per ml) illustrates that the nanoarray-based assay can exceed the limit of detection of conventional enzyme-linked immunosorbent assay (ELISA)- based immunoassays (5 pg per ml of plasma) by more than 1000-fold. Arrays of proteins with well-defined feature size and spacing are important for studying surface-cellular interactions 1,2 and detecting specific biomacromolecules. 3-5 Recently, there have been significant developments in the use of nanolitho- graphy techniques for patterning surfaces with proteins on the submicrometer length scale. 6-13 Dip-pen nanolithography (DPN) is one technique that has shown particular promise in this area, allowing one to prepare standardized multi- component arrays of biomolecules that can retain their biorecognition properties once transferred to a surface. 14-22 A key issue pertains to the potential of such nanostructures in medical diagnostics, and at present it is unclear what advantages such structures will offer for clinical applica- tions. 23 In principle, one can use smaller sample volumes and achieve higher sensitivity due to the small size of the entire array and the individual features that comprise the array. Such improved detection systems would enable the diagnosis of infection with HIV-1 in the setting of mother- to-child transmission, for example, where small sample volumes and the presence of immune complexes consisting of passively transferred maternal antibodies and HIV-1 antigen present diagnostic challenges. Polymerase chain reaction (PCR) and other forms of target amplification have enabled the development of powerful tools for detecting and quantifying HIV-1 nucleic acid targets for clinical diagnosis and prognosis. Though simpler to perform, conventional immunoassays for HIV-1 Gag p24 cannot achieve this level of sensitivity. Herein, we show how DPN-fabricated nanoar- rays of modified monoclonal antibodies against HIV-1 p24 can be used to detect the protein in plasma samples using gold nanoparticles modified with polyclonal anti-p24 IgG as probes in a three-component sandwich (Scheme 1). These data illustrate our capability to detect and measure HIV-1 p24 antigen by a nanoarray-based assay that exceeds the limit of detection of conventional ELISA-based immunoassays and provides a level of sensitivity comparable to a PCR-based assay without target amplification. In a typical experiment, a nanoarray for the HIV-1 immunoassay was fabricated by initially patterning 16- mercaptohexadecanoic acid (MHA) into dot features as small as 60 nm (10 × 10 spot array) on a gold thin film using DPN. The large spacing between features improved our ability to locate the original pattern after reaction with biomolecules or gold nanoparticle probes. At pH 7.4, the * Corresponding authors. E-mail: camirkin@chem.northwestern.edu and s-wolinsky@northwestern.edu. † Department of Chemistry and The Institute for Nanotechnology. ‡ The Feinberg School of Medicine. NANO LETTERS 2004 Vol. 4, No. 10 1869-1872 10.1021/nl049002y CCC: $27.50 © 2004 American Chemical Society Published on Web 09/09/2004