Simultaneous Detection Of Multiple Analytes Research Articles

Unlocking multi-mode sensing potential: Phosphorus-doped graphitic carbon nitride quantum dots for Ag+, ciprofloxacin, and riboflavin analysis in environment and food matrices

The simultaneous detection of multiple analytes through a single fluorescence sensor is highly attractive. In this study, phosphorus-doped graphitic carbon nitride quantum dots (P-CNQDs) were developed, achieving multi-mode sensing through three distinct response mechanisms. The preparation involved using melamine as the carbon and nitrogen source and ammonium dihydrogen phosphate as the phosphorus source. Uniform and narrowly distributed P-CNQDs were successfully synthesized through chemical oxidation and hydrothermal methods, with an average size of 2.4 nm. These unique P-CNQDs exhibited fluorescence quenching through photo-induced electron transfer (PET) in response to Ag+. Additionally, the formation of hydrogen bonds and coordination interactions between P-CNQDs-Ag+ and ciprofloxacin (CIP) led to a pronounced fluorescence response to CIP by the chelation enhanced fluorescence (CHEF) mechanism. Furthermore, leveraging the principle of fluorescence resonance energy transfer (FRET), P-CNQDs-CIP served as a ratio fluorescence sensor for riboflavin (RF), enabling ultra-sensitive detection of RF. The combination of PET, CHEF, and FRET response mechanisms successfully facilitated multi-mode sensing for Ag+, CIP, and RF. The detection ranges were 0.05–100 μM, 0.002–2 μM, and 0.05–60 μM, with corresponding lowest detection limits of 17.1 nM, 1.1 nM, and 29.2 nM, respectively. This versatile sensor has been effectively applied to real samples, including the detection of river water and vitamin B2 tablets.

Synthesis of eco-friendly multifunctional dextran microbeads for multiplexed assays

There has been an increasing demand for simultaneous detection of multiple analytes in one sample. Microbead-based platforms have been developed for multiplexed assays. However, most of the microbeads are made of non-biodegradable synthetic polymers, leading to environmental and human health concerns. In this study, we developed an environmentally friendly dextran microbeads as a new type of multi-analyte assay platform. Biodegradable dextran was utilized as the primary material. Highly uniform magnetic dextran microspheres were successfully synthesized using the Shirasu porous glass (SPG) membrane emulsification technique. To enhance the amount of surface functional groups for ligand conjugation, we coated the dextran microbeads with a layer of dendrimers via a simple electrostatic adsorption process. Subsequently, a unique and efficient click chemistry coupling technique was developed for the fluorescence encoding of the microspheres, enabling multiplexed detection. The dextran microbeads were tested for 3-plex cytokine analysis, and exhibited excellent biocompatibility, stable coding signals, low background noise and high sensitivity.

Three species multiplexing of fluorescent dyes and gold nanoclusters recovered with fluorescence lifetime correlation spectroscopy

Biosensor applications often require the simultaneous detection of multiple analytes, with a clear need to go beyond the traditional multiplexing relying on distinct fluorescent dyes across the visible spectrum. Fluorescence lifetime correlation spectroscopy (FLCS) is a powerful approach taking advantage of the fluorescence lifetime information to separate the contributions of different fluorescent species with overlapping emission spectra. However, so far FLCS detection has been demonstrated only on binary mixtures of two fluorescent dyes, limiting its multiplexing capabilities. Here, we report the first quantitative FLCS measurements within a ternary mixture composed of three different fluorescent emitters with near-identical emission spectra. Two organic fluorescent dyes, Alexa Fluor 647 and CF640R, are combined with water-soluble Au18(SG)14 gold nanoclusters. Our experimental data establish that FLCS allows to accurately determine individual concentrations within intricate ternary mixtures. Another major aspect of interest concerns the assessment of the suitability of gold nanoclusters for FLCS multiplexing applications. With their microsecond lifetime and stable emission characteristics, gold nanoclusters add a valuable new aspect to the array of FLCS probes. Extending FLCS multiplexing beyond binary mixtures paves the way for further progress in the simultaneous highly parallel biosensing of multiple species.

Simultaneous SERS Detection of Multiple Amino Acids Using ZIF-8@AuNPs as Substrate: Classified with 1D Convolutional Neural Network

Simultaneous SERS Detection of Multiple Amino Acids Using ZIF-8@AuNPs as Substrate: Classified with 1D Convolutional Neural Network

Multiplex Aptamer-Based Fluorescence Assay Using Magnetism-Encoded Nanoparticles for Simultaneous Detection of Multiple Pathogenic Bacteria.

Multiplex assay has emerged as a robust and versatile method for the simultaneous detection of multiple analytes in a single test. However, challenges in terms of poor accuracy and complexity remained. In this work, we developed a multiplex aptamer-based fluorescence assay using magnetism-encoded nanoparticles for the simultaneous detection of multiple pathogenic bacteria. The encapsulation of different amounts of Fe3O4 nanoparticles in zeolitic imidazolate framework-90 (ZIF-90) leads to the formation of Fe3O4@ZIF-90 (FZ) composites with distinct magnetism strengths. By functionalizing a specific aptamer on the surface of the FZ composites, target bacteria can be specifically and precisely separated from a mixed sample in a sequential manner. This property allows for the simultaneous quantitative analysis of multiple target bacteria by using a single-color fluorescence label, thereby resulting in minimal spectral crosstalk interference and improved accuracy. The successful determination of multiple bacteria in contaminated milk samples demonstrates the applicability of this multiplex assay in complex biological matrices. Compared to conventional multiplex fluorescence assays, this approach offers distinct advantages of simplicity, efficiency, and implementation. We believe that this study can provide valuable insights into the development of the multiplex assay while introducing a new method for the simultaneous detection of multiple bacteria.

A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

Electrochemical Biosensor Arrays for Multiple Analyte Detection

AbstractThe simultaneous detection of multiple analytes using electrochemical biosensor arrays has demonstrated significant potential in various fields, including medical diagnostics. These platforms are designed to be low‐cost, easy to use, and offer fast detection of a range of molecules. This concept article discusses the components necessary to achieve multiplexing with electrochemical biosensor arrays. The various methods used to fabricate electrode arrays of different types are discussed. Furthermore, the methods for the selective immobilization of multiple different bioreceptors onto individual electrodes within the array, a crucial step essential for conferring specificity to the analytical processes, are presented with relevant examples. We have focused on enzymes, antibodies and aptamers as examples of bioreceptors that have been deployed in various multianalyte electrochemical detection platforms. Finally, we discuss the key challenges associated with their application for the analysis of real samples and provide a future outlook on possible strategies that can be used to overcome these challenges.

Ultra-stable silver nanoplates: efficient and versatile colorimetric reporters for dipstick assays.

Noble metal anisotropic nanostructures, such as silver nanoplates (AgNPls), are interesting because they possess enhanced plasmonic properties compared to their spherical counterparts: increased extinction coefficient and tunable maximum of absorption wavelength. However, their use for biosensing application is limited as these structures are intrinsically unstable and, to maintain the anisotropic structure, a coating protecting the metallic surface is required. In this work, we report on the capacity of a thin but robust coating based on calixarene-diazonium salts to maintain the structure anisotropy of silver nanoplates in conditions in which traditionally used coatings fail. We synthesized AgNPls of various sizes and coated them with two different calixarenes, differing by the functional groups attached to their small rim. After characterization of the efficiency of the ligand exchange process between the initial citrate anions and the calixarenes, the chemical and colloidal stabilities of the resulting calixarene-coated AgNPls were compared to citrate-capped AgNPls. A radical improvement of the lifetime of the material from 1 day for AgNPls coated with citrate to more than 900 days for calixarene-coated AgNPls, as well as the stability in acidic conditions, phosphate saline buffer (PBS) or biofluid, were observed. Benefiting from this exceptional robustness, calixarene-coated AgNPls were exploited to design dipstick assays. Rabbit immunoglobulin G (IgG) detection was developed first as proof-of-concept. The optimal system was then used for the detection of Anti-SARS-CoV-2 IgG. In both cases, a picomolar limit of detection (LOD) was achieved as well as the detection in 100% of pooled human plasma. This sensitivity competes with that of ELISA and is better than the one previously obtained with gold or even silver nanospheres for the same target and in similar conditions. Finally, the wide range of colors provided by the AgNPls allowed the design of a multicolor multiplex assay for the simultaneous detection of multiple analytes.

Highly Sensitive Electrochemical Immunoassay Using Signal Amplification of the Coagulation Cascade

Here, we report a highly sensitive immunoassay for human immunoglobulin G (IgG) that uses signal amplification of the coagulation cascade. Z-Phe-Pro-Lys-p-nitroaniline (FPK-pNA) was used as a substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, pNA is liberated from FPK-pNA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that pNA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK-pNA and pNA. Characterization of the reactivity of thrombin toward FPK-pNA revealed that thrombin efficiently reacted with FPK-pNA. Subsequent characterization of factor XIa activity of factor XIa-labeled antibody revealed that factor XIa was not inactivated during labeling. Finally, a coagulation cascade-based immunoassay for human IgG was performed using a factor XIa-labeled antibody on magnetic beads. The limit of detection for human IgG was 5.0 pg/mL (33 fM) indicating that the coagulation cascade can amplify the immunoassay sensitivity compared to immunoassay using a thrombin-labeled antibody as a condition without a coagulation cascade. Coagulation cascade-based immunoassay was also highly selective. In the near future, we will report a highly sensitive immunoassay for the simultaneous detection of multiple analytes using a coagulation cascade-based immunoassay and Limulus amebocyte lysate reaction-based immunoassay we previously reported.

Dual-channel step multimode fiber SPR sensor based on sawtooth structure

The fiber SPR sensor with a single sensing channel can not detect multiple analytes at the same time and introduce the reference channel to eliminate nonspecific interference. Hereby we proposed a sawtooth dual-channel fiber SPR sensor based on WDM technology. By CO2 laser, multiple V-shaped grooves are processed on the step multimode fiber to form a sawtooth structure. The first stage of the sensor was fabricated by coating gold film on the sawtooth area, and the second stage was fabricated by coating gold film on the cladding area 2 cm behind the sawtooth area. The sensitivity and dynamic range of the two channels are 3114.86 nm/RIU and 1584.23 nm/RIU, 719.93 nm-854.2 nm, and 584.24 nm-667.62 nm, respectively. The proposed fiber SPR sensor employs the sawtooth structure to adjust the SPR resonance wavelength, so as to realize dual-channel sensing, which is of significance in the fields of simultaneous detection of multiple analytes and temperature compensation of reference channels.

Standard slot waveguide and double hybrid plasmonic waveguide configurations for enhanced evanescent field absorption methane gas sensing

Herein, a numerical study on standard slot waveguide and double hybrid plasmonic waveguide based on a silicon-on-insulator platform is presented. The geometric parameters of both the waveguides are optimized for the operational wavelength of 3.39 μm (absorption line of methane gas) to obtain the maximum evanescent field ratio (EFR). By utilizing Lambert-Beer’s law, the gas sensing capability of both the waveguides is determined. It is found out that both the waveguides of length 100 μm offer high EFR resulting in the 3dB decay of the propagating mode power for the methane gas concentration of 20-22 % in the chamber. The study provides the foundation for the practical realization of compact and highly sensitive gas sensors. Full Text: PDF ReferencesJ.Y. Yo, Y.S. Kwon, J.W. Lee, J.S. Park, B.H. Rho, W. II. Choi. "Acute Respiratory Distress Due to Methane Inhalation", Tuberculosis and Respiratory Diseases 74, 120-123 (2013). CrossRef M. A. Butt, S. A. Degtyarev, S. N. Khonina and N. L. Kazanskiy. "An evanescent field absorption gas sensor at mid-IR 3.39 μm wavelength", Journal of Modern Optics 64, 1892-1897 (2017). CrossRef M. A. Butt, S. N. Khonina and N. L. Kazanskiy. "Modelling of Rib channel waveguides based on silicon-on-sapphire at 4.67 μm wavelength for evanescent field gas absorption sensor", Optik 168, 692-697, (2018). CrossRef M. A. Butt, S. N. Khonina and S. N. Kazanskiy. "Silicon on silicon dioxide slot waveguide evanescent field gas absorption sensor", Journal of Modern Optics 65, 174-178, (2017). CrossRef S. N. Khonina, N. L. Kazanskiy and M. A. Butt. "Evanescent Field Ratio Enhancement of a Modified Ridge Waveguide Structure for Methane Gas Sensing Application", IEEE Sensors Journal 20, 8469-8476 (2020). CrossRef M.Vlk, A. Datta, S. Alberti, H.D. Yallew, V. Mittal, G. S. Murugan, J. Jagerska. "Extraordinary evanescent field confinement waveguide sensor for mid-infrared trace gas spectroscopy", Light: Science & Applications 10, 26 (2021). CrossRef M.A. Butt, S.N. Khonina, N.L. Kazankiy. "Enhancement of evanescent field ratio in a silicon strip waveguide by incorporating a thin metal film", Laser Physics 29, 076202 (2019). CrossRef M. A. Butt, N. L. Kazanskiy and S. N. Khonina, "Highly integrated plasmonic sensor design for the simultaneous detection of multiple analytes", Current Applied Physics 20, 1274-1280 (2020). CrossRef T. Milde, M. Hoppe, H. Tatenguem, C. Assmann, W. Schade, J. Sacher. "Comparison of the spectral excitation behavior of methane according to InP, GaSb, IC, and QC lasers as excitation source by sensor applications", Applied Optics 58, C84 (2019). CrossRef N. L. Kazanskiy, S.N. Khonina, M.A. Butt. "Polarization-Insensitive Hybrid Plasmonic Waveguide Design for Evanescent Field Absorption Gas Sensor", Photonic Sensors 11, 279-290 (2021). CrossRef D. Popa, F. Udrea. "Towards Integrated Mid-Infrared Gas Sensors", Sensors 19, 2076 (2019). CrossRef S-W. Kang, K. Sasaki, H. Minamitani. "Sensitivity analysis of a thin-film optical waveguide biochemical sensor using evanescent field absorption", Applied Optics 32, 3544-3549 (1993). CrossRef

Photochemical synthesis of Au nanostars on PMMA films by ethanol action as flexible SERS substrates for in-situ detection of antibiotics on curved surfaces

• Photochemical fabrication of Au NSs by the action of ethanol has been demonstrated. • Au NSs are employed directly on the flexible PMMA substrate for SERS application. • High enhancement factors about 10 9 are achieved for the antibiotics. • Effective multiplex SERS detection towards CPX and CAP is presented. • In-situ detection of antibiotics in chicken-wing samples was performed. Enormous Raman signal enhancement using gold nanostars (Au NSs) has attracted great attention due to the excitation of strong electric field on the nanostar tips. The synthetic methods of Au NSs mainly utilize specialized shape-directing agents, such as surfactants or polymers, to assist the tip formation. However, these agents passivate nanostar surfaces and weaken the interaction between Au NSs and analyte molecules. Herein, we report the synthesis of Au NSs with clean surface by the action of ethanol through a facile photoreduction process. The growth mechanism of Au NSs is elucidated and discussed with the action of ethanol as a polar protic solvent. The synthesis process of Au NSs is employed directly on poly(methyl methacrylate) (PMMA) films for the application of surface-enhanced Raman spectroscopy (SERS). The proposed flexible Au-NSs/PMMA SERS substrate is characterized by the SERS detection of two antibiotics, ciprofloxacin (CPX) and chloramphenicol (CAP). The superior detection performance includes high sensitivity, high enhancement factor (2.03 × 10 9 ), low limit of detection (3.41 × 10 -11 M), superior multiplex detection ability, good mechanical stability, excellent uniformity and reproducibility (<7.32 %). These superior sensing properties can be attributed to a large number of hotspots formed by diverse nanostructures at different positions of Au NSs on the Au-NSs/PMMA SERS substrate. The multiplex SERS detection ability is also explored for the simultaneous detection of multiple analytes. The transparent and flexible nature of the Au-NSs/PMMA SERS substrate enables the in-situ, real-time detection of CPX and CAP in chicken-wing samples by using the fiber-coupled Raman probe with the low LODs. The reported flexible SERS substrate opens the way to improve the practical detection of various antibiotics for the worldwide food safety issues.

Multiplex DNA Walking Machines for Lung Cancer-Associated miRNAs.

Biomimetic DNA walking machines have gained great success in scrutinizing the microscopic world and sensitive biosensing of disease biomarkers. Despite superb achievements, the research on DNA walking machines for simultaneous detection of multiple analytes is still rare, while the design and realization of multiplexing are considered as an important bottleneck. The multiplex detection of biomarkers can not only improve the specificity of bioassays but also avoid the squander of valuable biological specimens. Herein, we reported multiplex three-dimensional (3D) DNA walking machines based on high-resolution inductively coupled plasma mass spectrometry (HR-ICPMS) for lung cancer-associated miRNA detection. In the presence of lung cancer-associated target miRNAs (miR-21, miR-141, and miR-125b), DNA walking machines were stimulated and operated to liberate a large number of lanthanide elements (Tb, Ho, and Tm), and then the signals were collected simultaneously by HR-ICPMS. The recovery test of target miRNAs in human serum and the simultaneous monitoring experiment of three miRNAs in human lung cancer cell line (A549) and normal cell line (HBE) specimens display satisfactory analysis capabilities for complex biological samples. Thanks to the vast potential of lanthanide tags and the modular design, the proposed bioassay might flexibly detect different miRNA combinations with corresponding sets of DNA walking machines to meet the requirements of various tasks.

Evolution of “On-Barcode” Luminescence Oxygen Channeling Immunoassay by Exploring the Barcode Structure and the Assay System

The multiplexed luminescence oxygen channeling immunoassay (multi-LOCI) platform we developed recently that combines conventional LOCI and suspension array technology is capable of realizing facile “mix-and-measure” multiplexed assays without tedious washing steps. However, previous work lacks comprehensive studies of the structure–performance relationship of the host–guest-structured barcode, which may obstruct the evolution and further translation of this exciting new technology to practical applications. Accordingly, this work revealed that polyelectrolyte interlayers played a crucial role in tuning the packing density of guest acceptor beads (ABs). More interestingly, we noticed that “sparse” barcodes (barcodes with low ABs packing density) exhibited comparable assay performance with “compact” ones (barcodes with high ABs packing density). The high robustness of barcodes allows for multi-LOCI to be a more universal and flexible assay platform. Furthermore, through optimization of the assay system including the laser power, as well as the concentrations of donor beads and biotinylated detection antibodies, the multi-LOCI platform showed a significant improvement in sensitivity compared with our previous work, with the limit of detection decreasing to as low as ca. 1 pg/mL. Impressively, multi-LOCI that enabled simultaneous detection of multiple analytes exhibited comparable sensitivity with the classical single-plexed LOCI, due to the ingenious structural design of the multi-LOCI barcode and the unique “on-barcode” assay format.

Imaging of Plant Hormones with Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry.

Plant hormones can act in synergistic and antagonistic ways in response to biotic and abiotic stresses and during plant growth and development. Thus, a technique is needed to simultaneously determine the distribution and concentration of several plant hormones. A relatively new technology, mass spectrometry imaging (MSI), enables the direct mapping and imaging of biomolecules on tissue sections. MSI permits simultaneous detection of multiple analytes on a single section of plant tissue, even in the absence of target-specific markers such as antibodies. Recently, MSI has been used to localize multiple, small molecule (m/z<500) plant hormones by the nanoparticle-assisted laser desorption/ionization (Nano-PALDI) mass spectrometry (MS) method. Here, we illustrate a technology for multiple-hormone imaging using Nano-PALDI MSI and discuss its potential in investigating the role of hormone signaling in plant development and stress responses.

Protocell arrays for simultaneous detection of diverse analytes

Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when done at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexed analyte sensing still typically faces the critical limitation of measuring only one type of molecule (e.g., small molecules or nucleic acids) per assay platform. Here, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS), producing membrane-less protocells containing transcription and translation machinery used for detection. We show that multiple protocells, each performing a distinct sensing reaction, can be arrayed in the same microwell to detect chemically diverse targets from the same sample. Furthermore, these protocell arrays are compatible with human biofluids, maintain function after lyophilization and rehydration, and can produce visually interpretable readouts, illustrating this platform’s potential as a minimal-equipment, field-deployable, multi-analyte detection tool.

Multi-spiked silver stars for ultrasensitive and multiplexed SERS detection of analytes

We report a facile synthesis procedure of multi-spiked star-shaped silver structures with sharp morphological features and broad plasmonic absorption for the fabrication of surface-enhanced Raman scattering (SERS) substrates. The multi-branched morphology of silver stars (AgSs) provides copious hot spots on SERS-active substrates to detect ultra-low concentrations of probe molecules. Significant Raman intensity enhancement factors of the order of ∼109 were obtained for the different analytes. The AgSs-based SERS-substrates show ultra-sensitive SERS performance for the detection of probe molecules rhodamine B, methylene blue, thiram, and phosmet with detectabilities 2.6 × 10−16, 1.97 × 10−17, 1.78 × 10−18, and 4.08 × 10−14 M concentration, respectively. The improved SERS sensitivity originated from the additional contribution of chemical enhancement, corroborated by the appearance of new Raman bands in the SERS spectrum. For practical applications, SERS based molecule detection requires high sensitivity, specificity, uniformity, and reproducibility of the SERS signal. We demonstrate the simultaneous detection of multiple analytes, with high specificity, from the complex mixture of molecules with different compositions. The fabricated SERS substrates provide excellent signal uniformity and reproducibility with ∼10% relative standard deviation for the multiple substrates. The real-life application of AgSs-based SERS substrates with the spiked apple juice and ITO-coated PET films without pre-processing is also demonstrated.

Multivariate Analysis of a Cobalt Octaethyl Porphyrin-Functionalized SWNT Microsensor Device for Selective and Simultaneous Detection of Multiple Analytes

Multivariate Analysis of a Cobalt Octaethyl Porphyrin-Functionalized SWNT Microsensor Device for Selective and Simultaneous Detection of Multiple Analytes

Microfluidics-Based Plasmonic Biosensing System Based on Patterned Plasmonic Nanostructure Arrays.

This review aims to summarize the recent advances and progress of plasmonic biosensors based on patterned plasmonic nanostructure arrays that are integrated with microfluidic chips for various biomedical detection applications. The plasmonic biosensors have made rapid progress in miniaturization sensors with greatly enhanced performance through the continuous advances in plasmon resonance techniques such as surface plasmon resonance (SPR) and localized SPR (LSPR)-based refractive index sensing, SPR imaging (SPRi), and surface-enhanced Raman scattering (SERS). Meanwhile, microfluidic integration promotes multiplexing opportunities for the plasmonic biosensors in the simultaneous detection of multiple analytes. Particularly, different types of microfluidic-integrated plasmonic biosensor systems based on versatile patterned plasmonic nanostructured arrays were reviewed comprehensively, including their methods and relevant typical works. The microfluidics-based plasmonic biosensors provide a high-throughput platform for the biochemical molecular analysis with the advantages such as ultra-high sensitivity, label-free, and real time performance; thus, they continue to benefit the existing and emerging applications of biomedical studies, chemical analyses, and point-of-care diagnostics.

Multiple-analyte mass detection in a coupled microresonator array

Resonant sensors using coupled micro-cantilever arrays have found wide applications in the ultrasensitive mass detection of biomolecules and chemical analytes. Experimental observations indicate that a target mass deposited on one of the cantilevers can be detected by measuring the change in resonant frequencies or in eigenmodes. Analytical works have studied eigenvalue and eigenmode sensitivities, but for a single analyte only. Since a resonator array consists of several cantilevers, it offers an opportunity for the simultaneous detection of multiple analytes. However, multiple-analyte mass detection has not been investigated so far. In this paper, an analytical foundation for the detection of multiple analytes, through the measurement of eigenvalue shifts, is developed using matrix perturbation theory. The formulation presents a system of over-determined linear equations in terms of unknown analyte masses. A novel approach based on solving the equations in a least square sense is proposed and it is shown that it gives far better estimation accuracy than using a subset of equations for direct solution. The approach is demonstrated through numerical simulation for a typical three-cantilever array for the detection of two analyte masses. Estimation errors are studied for a range of analyte masses and presented in the form of an error surface. The effect of interconnection stiffness and array size on estimation error is also investigated. The robustness of the method is further tested against manufacturing variations and it is shown that an envelope guideline of maximum estimation error can be constructed for the user.