Optofluidic wavelength division multiplexing for single-virus detection

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context––the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine.The ability to overlay multiple electromagnetic waves in the same physical space by virtue of linear superposition is arguably at the root of modern communication as we know it. Originally implemented in the radiofrequency regime, this wavelength division multiplexing (WDM) principle was transferred to optical wavelengths in the visible and near-infrared range, which can be carried by a single, low-loss silica fiber (1). Available in both coarse and dense varieties, terabits of data are now shuttled between a source and their destination using anywhere from 4 to over 100 wavelengths (2, 3). Here, we transfer the WDM principle from data communications into a different realm, that of chip-based biomedical analysis, where much can be gained by superimposing multiple colors in an optical waveguide, albeit for different reasons.First, one of the key requirements for diagnostic test panels, aside from high sensitivity and specificity, is the ability to multiplex, i.e., detect and identify multiple biomarkers simultaneously. A standard influenza test, for example, simultaneously screens for eight pathogen types, enabling differential diagnosis of diseases with similar early symptoms (www.questdiagnostics.com/testcenter/testguide.action?dc=TS_RespVirusPanel). Current gold-standard techniques for nucleic acid and protein detection such as PCR and ELISA use fluorescent organic dyes as a means of signal reporting (4). Optical detection, by fluorescence or “label-free,” is extremely sensitive, allowing for single-molecule and even single-dye detection under appropriate conditions (5–11). The availability of over a dozen dyes across the visible spectrum opens the door to implementing the desired multiplexing capability with multiple dyes, i.e., spectral channels (12–14). Secondly, diagnostic tests are rapidly transitioning toward integrated laboratory-on-chip platforms on which small volumes of biological or chemical samples can be rapidly analyzed. The WDM principle of routing all spectral channels through the same physical space is, therefore, ideal for increasing compactness of an analytic device. Finally, chip-scale integration has recently been advanced by the advent of optofluidic devices in which both fluidic and optical components are miniaturized in the same system (15–17).Here, we implement WDM on an optofluidic platform for on-chip analysis of single influenza viruses. In place of silica fiber as the physical carrier, we create a single waveguide structure that combines multiple spectral channels for fluorescence excitation of biological targets. Instead of temporally modulating each channel to transport information, we use this waveguide to produce wavelength-dependent spatial patterns in an intersecting fluidic channel. The spatial encoding of spectral information then allows for direct identification of multiple labeled targets with extremely high sensitivity and fidelity. The technique is demonstrated in two implementations for direct counting and identification of individual virus particles from three different influenza A subtypes––H1N1, H2N2, and H3N2––at clinically relevant concentrations.