Microarray
A microarray is a high-throughput laboratory technique in molecular biology that enables the simultaneous analysis of thousands to millions of biomolecules, such as DNA, RNA, proteins, or antibodies, by immobilizing them as probes on a solid substrate like a glass slide or silicon chip and detecting interactions with labeled target samples through hybridization or binding assays.[1][2] Developed in the mid-1990s, microarray technology revolutionized genomics by providing a cost-effective alternative to full DNA sequencing for large-scale studies, allowing researchers to measure gene expression levels, identify genetic variations such as single nucleotide polymorphisms (SNPs), and detect mutations associated with diseases.[3][4] The process typically involves spotting or synthesizing probes in a grid pattern on the substrate, hybridizing fluorescently labeled sample nucleic acids or proteins to complementary probes, and scanning the resulting fluorescence intensity to quantify binding, which reveals relative abundance or presence of targets.[5][3] There are several types of microarrays, broadly categorized by the biomolecules involved. DNA microarrays, the most common, include spotted arrays where pre-synthesized DNA fragments are deposited robotically on coated glass slides, in situ synthesized arrays using photolithography or inkjet printing to build oligonucleotides directly on the surface, and bead-based arrays with DNA-attached microspheres in etched wells.[4] Protein microarrays, on the other hand, immobilize proteins, peptides, antibodies, or glycans to study protein interactions, biomarker discovery, or immune responses, often employing functional, analytical, or reverse-phase formats for applications in diagnostics and drug development.[2] Key applications of microarray technology span research and clinical settings. In genomics, DNA microarrays facilitate genome-wide association studies (GWAS) to link genetic variants with diseases like breast cancer or diabetes, measure differential gene expression between healthy and diseased tissues, and support prenatal diagnosis of chromosomal abnormalities via chromosomal microarray analysis (CMA).[1][6] Protein and antibody microarrays aid in pathogen detection, such as identifying SARS-CoV-2 variants or multiple swine viruses with high sensitivity (e.g., down to 10² copies/µL).[2] Additionally, they contribute to personalized medicine by enabling genotyping for pharmacogenomics and monitoring immune responses in vaccine development.[3][2] Despite their versatility, microarrays have limitations, including reliance on prior knowledge of probe sequences, potential cross-hybridization leading to false positives, and lower resolution compared to next-generation sequencing (NGS).[4] As of 2025, while DNA microarrays for genotyping and chromosomal analysis remain widely used in clinical diagnostics due to their speed and affordability, gene expression profiling has increasingly shifted to RNA sequencing for its unbiased detection and higher dynamic range, though microarrays continue to evolve with improvements in fabrication, such as 3D structures and label-free detection methods like surface plasmon resonance imaging (SPRi), alongside recent advancements including AI-enhanced analysis and integrated genotyping devices.[2][7][8][9]Overview
Definition and Principles
A microarray is a high-throughput analytical platform consisting of a solid support with an ordered array of microscopic spots, each containing immobilized biomolecular probes such as DNA oligonucleotides, cDNA fragments, or proteins, arranged in a grid-like format to enable the simultaneous detection and quantification of multiple target analytes through specific binding interactions.[10] These probes are designed to capture complementary targets from a sample, such as nucleic acids or proteins, facilitating parallel analysis of thousands to millions of interactions in a single experiment.[1] This technology extends beyond nucleic acids to include protein microarrays, where capture molecules like antibodies or antigens are spotted to study protein-protein, protein-DNA, or enzyme-substrate interactions.[11] The fundamental principles of microarray operation rely on the immobilization of probes onto a substrate, followed by the application of a labeled target sample, selective binding (e.g., hybridization for nucleic acids or affinity interactions for proteins), and detection of bound targets via signal intensity proportional to their abundance.[12] Substrates commonly include glass slides, silicon wafers, or plastic for their optical clarity and chemical stability, allowing precise spotting and scanning.[10] Binding specificity ensures that only complementary targets hybridize or associate with probes under controlled conditions like temperature and salt concentration, minimizing non-specific interactions, while washing steps remove unbound material to enhance signal-to-noise ratios.[13] Quantitative measurement typically involves fluorescence detection, where the intensity correlates with target concentration, enabling relative or absolute quantification.[12] Key components include probes, which are short, sequence-specific molecules (e.g., 20-100 nucleotides for DNA or antibodies for proteins) that serve as capture agents; targets, the analyte molecules (e.g., mRNA-derived cDNA or cellular proteins) from the biological sample; solid substrates for probe attachment; and labeling strategies, such as fluorescent dyes like Cy3 (green) and Cy5 (red) for dual-color detection in comparative assays.[10] These dyes are covalently linked to targets during preparation, allowing differential labeling of samples (e.g., control vs. experimental) for ratio-based analysis without needing identical probe amounts across arrays.[13] The general workflow begins with probe array creation through spotting or synthesis on the substrate, followed by target sample preparation involving extraction, amplification if needed, and fluorescent labeling (e.g., reverse transcription of RNA to cDNA for nucleic acid arrays).[12] Labeled targets are then incubated with the array under hybridization conditions to allow binding, excess targets are washed away, and the array is scanned using a laser confocal microscope to capture fluorescence signals from each spot, generating data for downstream analysis of target abundance.[13] This streamlined process supports high-density arrays, such as those monitoring expression of over 45 genes in early implementations, scalable to genome-wide levels.[13]Historical Development
The concept of microarrays was first introduced in 1983 by Tse Wen Chang for antibody microarrays, as described in a scientific publication and US Patent 4,376,110, laying the groundwork for multiplexed biomolecular analysis.[14] High-throughput DNA microarray technology originated in the early 1990s at Stanford University, where researchers Patrick O. Brown and Ronald W. Davis pioneered spotted complementary DNA (cDNA) arrays to study gene expression patterns on a genomic scale. These early arrays involved robotic spotting of DNA probes onto glass slides, enabling the simultaneous monitoring of thousands of genes through hybridization with labeled targets. The foundational work was detailed in a 1995 publication by Mark Schena, Dari Shalon, Davis, and Brown, which demonstrated quantitative gene expression analysis using fluorescent detection for 45 Arabidopsis thaliana genes, marking a significant advance over previous low-throughput methods like Northern blotting.[13] A key milestone occurred in 1995 when Affymetrix introduced high-density oligonucleotide arrays fabricated via photolithography, allowing for the in situ synthesis of up to hundreds of thousands of short DNA probes on a single chip. This light-directed approach, building on earlier concepts from Stephen Fodor's team, enabled precise spatial control and massively parallel analysis, particularly for genotyping and expression profiling. The technology's first commercial application was an HIV genotyping GeneChip in 1994. Affymetrix expanded to eukaryotic gene expression arrays in the mid-1990s, with early products like the yeast genome array released around 1997, solidifying the platform's impact.[15] During the late 1990s and 2000s, microarray technology expanded rapidly through commercialization by major companies, transitioning from academic prototypes to standardized platforms. Affymetrix dominated early oligo-based markets, while Agilent Technologies launched inkjet in situ synthesis arrays in 2000, offering customizable probe lengths and higher flexibility. Illumina introduced bead-based arrays in 2003, utilizing silica microbeads for genotyping and expression studies, which improved throughput and reduced costs. Protein microarrays also advanced during this period, with formats for antibody and antigen arrays emerging for proteomics applications in the early 2000s. This era also saw a shift from two-color microarray formats—common in spotted cDNA arrays for comparative hybridization—to single-color formats in commercial oligo and bead systems, enhancing data consistency and simplifying analysis.[16] In the 2010s, advancements integrated microarrays with next-generation sequencing (NGS) for hybrid workflows, such as microarray-based target capture to enrich specific genomic regions before sequencing, improving efficiency for clinical diagnostics and large-scale studies. Bead-based systems from Illumina further evolved, supporting multiplexed assays for epigenetics and copy number variation. By the early 2020s, innovations like nanomaterial-enhanced detection, including silver island films for metal-enhanced fluorescence, boosted sensitivity for low-abundance targets. The global microarrays market reached approximately USD 6.5 billion in 2024, fueled by post-COVID demand for diagnostic applications in infectious disease monitoring and personalized medicine.[9]Types of Microarrays
DNA Microarrays
DNA microarrays are specialized arrays designed for the analysis of nucleic acids, featuring probes that are typically short oligonucleotides of 25-70 bases or longer cDNA fragments, each representing specific genes, exons, or genomic regions. These probes are immobilized on a solid substrate, such as glass or silicon, allowing for high-density arrangements with up to millions of features per array, enabling parallel interrogation of thousands to entire genomes. Common formats include spotted arrays where pre-synthesized probes are deposited, in situ synthesized arrays built directly on the surface, and bead-based arrays using DNA-attached microspheres in etched wells for flexible genotyping applications.[4][17][18] The primary applications of DNA microarrays include gene expression profiling, where they measure mRNA levels to assess transcriptional activity across samples; comparative genomic hybridization (CGH), which detects copy number variations by comparing test and reference DNA; and single nucleotide polymorphism (SNP) genotyping, which identifies genetic variants for association studies. For instance, array CGH, pioneered in high-resolution formats, uses differentially labeled genomic DNA hybridized to arrays to reveal chromosomal gains or losses with precision down to kilobase scales, as demonstrated in early applications to cancer genomes.[4][19] Similarly, SNP genotyping via microarrays, with early assays targeting over 1,400 loci, has scaled to genome-wide coverage for population genetics and disease mapping.[4] DNA microarrays operate in two main formats: two-color systems, which involve competitive hybridization of two samples labeled with distinct fluorophores like Cy3 (green) and Cy5 (red) on the same array for direct ratio-based comparisons, and one-color systems, such as the Affymetrix GeneChip, where each sample is hybridized separately with a single label and data normalized across arrays. Whole-genome arrays cover the entire genome, while focused arrays target specific pathways or regions for cost-effective analysis.[4][20] A defining feature of DNA microarrays is their reliance on sequence-specific hybridization, governed by Watson-Crick base pairing, where target DNA or RNA binds complementarily to probes, though challenges like cross-hybridization—non-specific binding due to sequence similarity—can introduce errors, particularly in complex eukaryotic genomes, necessitating careful probe design and validation.[4] This principle builds on fundamental nucleic acid hybridization, allowing quantitative readout of binding affinity through fluorescence intensity.[4]Protein Microarrays
Protein microarrays, also known as protein chips, are high-throughput platforms where probes consisting of purified proteins, antibodies, or peptides are immobilized on a solid surface, such as glass slides or nitrocellulose membranes, to enable the simultaneous analysis of multiple protein interactions or activities.[21] Unlike nucleic acid-based arrays, these platforms rely on affinity-based capture rather than hybridization, with immobilization techniques ensuring the probes retain their native conformation for functional assays.[22] They are broadly classified into analytical arrays, which focus on detecting and quantifying protein analytes from complex samples, and functional arrays, which assess enzymatic activities or binding events, such as kinase-substrate interactions where thousands of substrates are screened against a kinase to map phosphorylation sites.[21] For instance, functional arrays have identified over 280 kinase substrates in human proteomes, highlighting their utility in signaling pathway elucidation.[22] Primary applications of protein microarrays include studying protein-protein interactions, profiling antibody specificities, and discovering biomarkers in serum or tissue samples. In protein-protein interaction studies, arrays featuring the entire yeast proteome (over 5,800 proteins) have detected interactions like those involving calmodulin with 30 targets, providing insights into cellular networks. Antibody profiling uses arrays to evaluate monoclonal antibody binding to immobilized antigens, ensuring specificity and reducing off-target effects in therapeutic development.[22] For biomarker discovery, arrays screen serum for autoantibodies, such as identifying three specific markers for autoimmune hepatitis diagnosis with high sensitivity.[22] Protein microarrays operate in two main formats: forward-phase and reverse-phase, each suited to different analytical needs.| Format | Description | Key Features and Examples |
|---|---|---|
| Forward-Phase | Purified probes (e.g., antibodies or proteins) are immobilized on the surface; complex samples (e.g., serum) are flowed over the array for capture. | High-throughput analyte detection; used for simultaneous profiling of multiple proteins in one sample, such as cytokine arrays for immune response analysis.[23] |
| Reverse-Phase | Complex samples (e.g., cell lysates or tissue extracts) are spotted onto the surface; specific probes (e.g., antibodies) are applied to detect targets. | Quantitative pathway analysis with minimal sample; applied to microdissected tumors to measure phosphorylated proteins in signaling cascades.[23] |