Densely Packed Microgoblet Laser Pairs for Cross‐Referenced Biomolecular Detection

Microgoblet laser pairs are presented for cross‐referenced on‐chip biomolecular sensing. Parallel readout of the microlasers facilitates effective mutual filtering of highly localized refractive index and temperature fluctuations in the analyte. Cross‐referenced detection of two different types of proteins and complete chemical transducer reconfiguration is demonstrated. Selective surface functionalization of the individual lasers with high spatial accuracy is achieved by aligned microcontact stamping.


DOI: 10.1002/advs.201500066
ring resonators with integrated waveguides, [ 7,8 ] no other chipbased WGM sensing platform capable of simultaneous signal processing of multiple microcavities has been demonstrated so far. However, this capability is crucial for real-world applications. It enables the compensation of sensing signal deteriorations originating, e.g., from localized density and temperature fl uctuations or from nonspecifi c molecular adsorption events. A sole temperature referencing can be performed with one single WGM transducer, but requires rather complex transducer preparation methods and well controlled high resolution WGM readout schemes. [ 1,15 ] Still, signal disturbances from refractive index fl uctuations or nonspecifi c molecular binding can solely be fi ltered out by the use of at least one additional reference transducer.
A promising solution to these restrictions are WGM microgoblet lasers fabricated out of polymers. [16][17][18][19][20] Here, the implementation of lasing dyes into the optical cavity results in active sensing elements, which can be probed and read out with very low alignment requirements via free-space optics. Since fabrication is performed photolithographically, the sensors can be realized cost-effi ciently and with very high device density and throughput on the wafer scale. [ 16 ] High lateral patterning accuracy allows a highly defi ned and reproducible geometrical design of the WGM cavities. Hence, defi ned cavity confi gurations can easily be realized, with each individual resonator exhibiting a distinct set of WGMs. Since the cavities can be structured very close to each other a more effi cient signal referencing is possible as even highly localized disturbances may be accounted for.
In this work we present a sensing platform for facile crossreferenced detection of multiple different molecules. We utilize high-Q microgoblet lasers fabricated from dye-doped poly(methyl methacrylate) (PMMA) [ 16 ] structured pairwise in close proximity to each other, allowing simultaneous free space optical pumping and readout. The individual laser cavities exhibit different radii and hence support mode sets with different free spectral ranges (FSR). Assignment of specifi c modes from the complex spectrum to each resonator is therefore straightforward. Utilizing the parallel readout of both resonators we discuss the cross-referencing capability in detail. By using one cavity as a reference at an assigned time period during the sensing experiment, signal disturbances arising from temperature and refractive index fl uctuations can mutually be compensated. Additionally, we show the detection of two different proteins with the very same microlaser pair. The microlasers are selectively surface functionalized with very high lateral accuracy with two different headgroup-modifi ed phospholipid inks by aligned microcontact stamping (AµCS). [ 18 ] One of the utilized inks comprises chemical end groups for reversible microfl uidic coupling and decoupling of linker-tagged proteins, enabling an in situ reconfi guration of the respective microlaser during the sensing experiment. By the subsequent detection of polyhistidine-tagged green fl uorescent protein (his-GFP) with the very same microlaser we show how referencing of the sensing signals results in a fi vefold improved signal reproducibility.
To ensure effi cient simultaneous pumping and readout, the individual microgoblet lasers of the microlaser pairs had a lateral distance of around 3 µm. Due to their different cavity radii, the microgoblets support sets of lasing modes with different FSRs.
Explicit assignment of the lasing peaks to the corresponding microlaser enables correlation of spectral shifts to molecular binding events occurring at the respective resonator circumference. The surface functionalization of the microlaser pairs is performed by AµCS, as depicted in Figure 1 a-d. As ink stamp pad a microscope glass slide is utilized, onto which phospholipid inks with different functional headgroups are deposited as laterally confi ned fi lms. Individual microlasers are selectively inked utilizing the interface areas between the laterally confi ned ink fi lms and the bare glass. Therefore the microlaser to be inked is aligned on the ink fi lm side and the second laser on the area of the bare glass of an ink spot interface. For all presented experiments the individual cavities of each microlaser pair are coated with different phospholipid inks to functionalize them with different molecular acceptors. The phospholipid transfer is typically limited to the circumference of the lasers as solely the goblet rims are brought into contact with the lipid ink spots (Figure 1 e,h).
Optical characterization is performed by using a laboratory spectrograph with a Peltier-cooled camera, equipped with a 2D charge-coupled device (CCD) pixel array ( Figure 2 a). Detailed simultaneous analysis of the lasing modes is realized by aligning the symmetry axis of the goblet pairs along the entrance slit of the spectrograph (Figure 2 a, inset picture). Within any biomolecular detection experiment, local temperature and refractive index fl uctuations of the analyte typically result in signifi cant sensor signal deterioration, in particular when the measurements are carried out outside of a controlled laboratory environment. For example, during point-of-care diagnostics ambient temperature can hardly be controlled. Here, incident sunlight or the body temperature of the operator or the patient may induce considerable signal deviations. Additionally, refractive index fl uctuations induced by the use of different analyte solutions or nonspecifi c molecule adsorption can further impair the sensing result.
To demonstrate the referencing capability of the microlaser pairs, we performed a molecular detection experiment mimicking these typical signal disturbances ( Figure 3 ). For this purpose, the small diameter microlaser was functionalized with a phospholipid ink consisting of 4 mol% Biotinyl Cap PE in 1,2-dioleoylsn -glycero-3-phosphocholine (DOPC) (later referred to as biotin ink) to obtain a sensor for streptavidin. To confi gure the large diameter laser as a passivated reference sensing element it was coated with pure DOPC and did therefore not provide any molecular binding sites. The goblet chip was then mounted into the microfl uidic chamber of the optical characterization setup. To induce temperature changes a Peltier element was glued to the metal frame of the microfl uidic chamber. Temperature monitoring was established by placing  The glass stamp pad comprises two different phospholipid ink fi lms and is glued upside down onto a custom made holder. A piezo-actuated six-axis translational stage is utilized for mechanical alignment of the microlaser chips relative to the stamp pad. The single microlasers are selectively coated by aligning the symmetry axis of the laser pair perpendicular to the respective lipid fi lm interface, with the respective targeted laser located on the inked and the second laser on the noninked side of the interface. The ink transfer procedure is visually controlled through the stamp pad via a CCD camera. e) In situ picture of the inking process prior stamp pad contact. f) Inking of the left goblet. A color change along the circumference indicates physical contact between ink fi lm and laser. g) SEM image of an inked microlaser pair. h) Close-up picture of the boxed area in (g) with lipid ink functionalization demarked by false color. The phospholipids are clearly limited to the circumferences of the lasers. a type K thermo couple temperature sensor into close vicinity to the chip. Prior to the sensing experiment the functionalized goblet pair was kept in a 0.5% bovine serum albumin (BSA) solution for 30 min to further suppress unspecifi c binding. At the beginning of the streptavidin detection the lasers were immersed in pure water at room temperature. The streptavidin (12 µg mL −1 ) injected after 1 min was diluted in PBS, so that the lasing modes of both lasers experienced an abrupt redshift due to the slightly higher refractive index of the PBS (Δ n = +0.0017). During the streptavidin incubation the temperature of the analyte was increased fi rst by +0.1 K at time point 12 min and then by additional +1 K at 14.5 min, to simulate arbitrary temperature drifts. At 16.5 min the Peltier element was switched off, to let the analyte cool down back to room temperature. Finally, the fl uidic chamber was purged with water. Direct analysis of the temporal spectral lasing mode shifts of the biotinylated laser ( Figure 3 , blue curve) shows considerable signal disturbances induced by the temperature changes. While the signal of the reference goblet ( Figure 3 , green curve) shows analogous disturbances, it additionally reveals the refractive index change induced by the PBS, which is not intuitively visible by the sole investigation of the biotinylated laser's sensing signal.
To fi lter out all these signal disturbances, the signal of the reference goblet can be utilized as a straightforward "reference baseline." For this purpose, the temperature and refractive index sensitivities (temperature sensitivity (TS) and bulk refractive index sensitivity (BRIS)) of the individual microgoblets must be known. The fi ltering can then be performed by an accordingly weighted point-to-point subtraction of the reference goblet signal from the sensing goblet signal. In prior experiments, the BRIS of the individual microlasers was experimentally assessed by exposing unfunctionalized microlaser pairs to different concentrations of glycerol in water and evaluating the resulting spectral shifts of the lasing modes. The average BRIS of the small diameter laser modes in nm per refractive index unit (RIU) was 20.07 nm RIU −1 (standard deviation 0.52 nm RIU −1 ). For the large diameter laser the measured BRIS was 14.80 nm RIU −1 (standard deviation 0.20 nm RIU −1 ). To characterize the TS the temperature was increased in discrete steps while the goblet pairs were kept in water. The average TS of the small diameter laser modes was −14.18 pm K −1 (standard deviation 0.25 pm K −1 ). The large diameter laser TS was measured to −14.99 pm K −1 (standard deviation 0.27 pm K −1 ).
Since the BRIS and TS of the two microlasers are different, the raw signal of the reference laser Δ λ ref,raw has to be adequately weighted before it can be utilized for referencing. The weighted reference signal is then subtracted from the raw signal Δ λ sens,raw of the sensing laser to obtain the referenced signal Δ λ sens,corr,t    S sens and S ref are either the TS or BRIS of the sensing, and the referencing laser to be considered at a time, respectively; Δ t is the acquisition time; Δ λ 0 represents the fi rst acquisition data point for offset correction.
The referencing leads to signifi cant improvement of the biotinylated laser's sensing signal (Figure 3 , black curve). Besides the accurate fi ltering of the temperature induced signal contributions, also the obscure signal offset triggered by the refractive index of the PBS is compensated. The referenced sensor signal of the biotinylated laser now delivers the generally expected exponentially saturating streptavidin binding curve. [ 21 ] If both microlasers provide different types of binding sites, mutual signal fi ltering can be performed (cross-referencing). The cross-referenced detection of two different kinds of proteins is demonstrated in Figure 4 . Additionally, within this experiment a targeted reconfi guration of one of the sensor elements is performed. For this purpose, the small diameter laser was functionalized with 25 mol% 1,2-dioleoyl-sn-glycero-3-{[ N (5amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DOGS-NTA-Ni) in DOPC to decorate the microgoblet with nickel (Ni) chelating headgroup components for selective binding of his-tagged molecules. As Ni/his-tag binding is reversible this ink allows facile multiple binding and removal of his-tagged acceptor molecules, enabling an in situ sensor reconfi guration with different types of biorecognition elements. The large diameter microlaser was coated with the biotin ink. The molecular binding experiment reveals the high lateral accuracy of the surface functionalization by AµCS. Despite the short lateral distance of the single microlasers (≈3 µm), their individual lasing modes exclusively respond to the specifi c binding of the respective target molecules (streptavidin and his-GFP (green fl uorescent protein)) (Figure 4 a). Figure 4 b shows that the binding curves of both microlasers are superposed by nonspecifi c signal contributions, which can be attributed to temperature and refractive index changes induced by the fl uid injections. For example, the his-GFP bound to the Ni-chelated microlaser in a fi rst incubation round (from 1.5 to 6.5 min) is removed by a solution of imidazole in PBS (7.5 to 7.75 min) to re-avail the Nichelate end groups for a subsequent second binding round (9.25 to 14.25 min) (Figure 4 b, blue curve). During this reconfi guration process a considerable disturbance of the Ni-chelated microlaser signal can be observed. First, the superposed step function during the blueshift of the Ni-chelated microlaser signal is the result of the higher refractive index of the imidazole solution (Δ n = +0.0016, compared to the PBS buffer used for the his-GFP), as indicated by the peak in the biotinylated laser signal (Figure 4 b, green curve). Second, after the reconfi guration the biotinylated laser signal is offset by ≈4.8 pm, indicating a slight nonspecifi c adsorption of imidazole molecules to both microlasers. Furthermore, the temporally bluedrifting signal of the biotinylated laser during both GFP incubation rounds indicates slight temperature increase induced at each injection event. Due to the sign of the drifts refractive index disturbances based on nonspecifi c his-GFP adsorption can be excluded. This indicates the generally very good protein repulsion characteristics of the phospholipid inks already observed in previous publications. [ 18,22 ] As a result of all these disturbances, the sensing signal generated by the Ni-chelated microlaser during the fi rst and the second his-GFP binding round shows signifi cant differences. Besides the signal offset, the two binding curves saturate at signifi cantly different values (fi rst his-GFP incubation round: 71.86 pm; second incubation round: 83.61 pm). For each analyte incubation the nonbinding specifi c signals occurring at the laser with the respective incompatible molecular acceptors can be utilized once more as a reference for the signals of the matching laser.  The mutual referencing of the sensing signals leads to the fi ltered molecular binding signals for both microlasers (Figure 4 b, black curves). A general improvement of the detection result is intuitively visible by the now constant temporal sensing signals prior and after the respective streptavidin/his-GFP injections, indicating accurate correction of the temperature induced drifts. Furthermore, the refractive index disturbance as well as the signal offset triggered by the nonspecifi c imidazole adsorption has fully been compensated. Eventually, the binding curves of the Ni-chelated goblet of both his-GFP incubation rounds now show a reduced deviation from previously 16.4% to now 3.2% (fi rst his-GFP incubation round saturation: 73.46 pm; second incubation round: 75.79 pm). This indicates a signifi cantly improved signal reproducibility of about a factor of 5. Based on the spectral resolution of our optical characterization setup and the accuracy of the Lorentzian peak-fi tting the experimentally found 3 σ -confi dence interval of our central lasing mode wavelength determination is ±1 pm ( σ : standard deviation). From the two subsequently acquired his-GFP sensing signals we estimated an average detection limit of 159.9 ± 14.6 ng mL −1 for the GFP prior signal referencing. After the referencing the detection limit lies at 159.1 ± 5.0 ng mL −1 . While the absolute detection limit remains virtually unchanged, the confi dence interval of the detection limit is diminished from 9.1% to 3.1%. In our estimation we assumed a measuring time of 5 min for the GFP and 7 min for the streptavidin (as performed in Figure 4 ), respectively. Furthermore, we assumed the sensing signals to scale linearly with protein concentration. [ 21 ] For the streptavidin we deduced a detection limit of approximately 133.9 ± 4.2 ng mL −1 from the referenced signal curve shown in Figure 4 . While most types of the signal deviations could explicitly be defi ned by visual inspection of the two lasers signals, some deviations can be interpreted differently. The approximate 3.5 pm redshift found in the Nichelated microlaser (now acting as the reference sensor) during streptavidin incubation (15.5 to 22.5 min) was inferred to be originating from slight refractive index disturbance due to nonspecifi c adsorption of streptavidin molecules. While this deduction leads to a satisfactory signal referencing, the drift may be alternatively interpreted as a result of a temperature decrease during incubation.
In conclusion, we have demonstrated a sensing platform for straightforward cross-referenced detection of multiple different molecules, based on the utilization of densely arranged WGM PMMA microgoblet laser pairs in the very same (microfl uidic) environment. The close proximity of the lasers enables parallel free space optical pumping and read-out, avoiding the alignment challenges of tapered fi ber coupling. We have shown that the simultaneously acquired spectral signals of the individual lasers can be harnessed for facile but effective mutual referencing. This allows compensation of parasitic signal disturbances originating from temperature and refractive index changes. This is a unique feature not readily available for other single WGM structures, where, e.g., sub-micrometer sized stray particles have to be introduced with high lateral precision to establish a pure temperature referencing by scattering induced mode splitting. [ 15 ] Additionally, our approach supersedes the very high resolution WGM detection schemes mandatory for resonance discrimination. [ 1 ] Furthermore, we have demonstrated the multiplexing capability by the detection of two different proteins. Besides microring-based structures with signifi cantly lower Q-factors, no other on-chip sensing platform allowing simultaneous interrogation of multiple WGM cavities has yet been demonstrated. We have shown that signal referencing leads to fi vefold increased signal reproducibility. We have shown AµCS to provide an unparalleled high lateral precision for selective and contamination-free functionalization of the individual lasers. We have demonstrated a novel approach for a facile complete chemical reconfi guration of WGM biosensing devices. In contrast to protocols re-availing the binding receptors for multiple detection of the same type of analyte molecule, [ 8 ] our approach opens up for the fi rst time the possibility to perform multiple completely independent binding experiments, e.g., by subsequently utilizing different acceptor molecules on the same WGM transducer. The reconfi guration is done using microfl uidics only and can be performed in closed lab-on-a-chip environments without requiring removal of the sample from the measurement system. [ 23 ] As his-and biotinconjugates are widespread in protein confi gurations a large variety of acceptor molecules is readily available. Our results are paving the way for the realization of complex multimolecular analyte experiments, e.g., with blood, which generally suffer from different analyte temperatures, local refractive index fl uctutions and nonspecifi c molecular adsorption. In this regard, the use of phospholipid inks as the surface functionalization might be particularly benefi cial, due to the known high protein repulsion. While we have already shown the good passivation capability against the proteins used within this manuscript, and on single microgoblet lasers against bovine serum albumin in our previous publication, [ 18 ] the sensing and signal referencing performance in more complex media like blood plasma still has to be confi rmed. Here, particularly lipid-based analyte components might interact with our surface functionalization, potentially resulting in signifi cant nonspecifi c signal contributions. Biosensor assemblies ("lipid gratings") of similar composition as in our present approach were already demonstrated to retain their geometry and specifi c sensing functionality in fetal calf serum in our lab. [ 22 ] We expect the current biosensor setup to perform equally well.
Our experiments presented here focus on the implementation of two lasers, allowing for the detection of multiple proteins in a sequential manner. To establish simultaneous detection larger laser arrays may be utilized availing at least one entirely passivated microlaser for signal referencing. While surface functionalization could be provided straightforward by multiplexed AµCS, [ 18 ] particularly larger microlaser arrays will require pump spot shaping for effective optical excitation of all microlasers. By the use of a microscope objective with lower magnifi cation and thus a larger fi eld of view, about ten microlasers may be simultaneously read out by our current optical characterization setup.
Within the presented experiments we assumed the signal disturbances to be mainly triggered by the instants of the fl uid injections. The type of disturbance for signal referencing was decided on by the user on visual assessment of the temporal signal progression of the two microlasers. The sensing signals were then referenced either for refractive index or for temperature disturbances at a time, not for both simultaneously. Accounting for signal disturbances at any time during measurement could be performed by an in situ (or post) analysis of the time progression www.MaterialsViews.com www.advancedscience.com of the reference and sensing laser signal. Based on the progression of the reference signal all possible correction factor combinations could then be applied to reconstruct the expected signal progression of the sensing signal. By this procedure different types of disturbances occurring simultaneously (e.g., nonspecifi c absorption-based refractive index disturbances superimposed by temperature drifts) would be accounted for.
The presented cross-referencing and microfl uidic reconfi guration capabilities along with the mass-fabrication compatibility [ 17 ] as well as facile detection schemes open up very promising perspectives for a utilization in portable lab-on-a-chip systems for point of care diagnostics. [ 20,24 ]

Experimental Section
Microgoblet Laser Fabrication : The microgoblet laser pairs were fabricated out of pyrromethene 597 (Radiant Dyes) doped PMMA. A 1.2 µm thick PMMA (PMMA 950k A6, MicroChem) layer was spun onto silicon wafers. Subsequently, arrays of microdisc pairs were structured into the PMMA layer by electron beam lithography. The pairs each comprised microdisks with radii R L = 25 µm and R S = 20 µm, with an edge-to-edge distance of 500 nm. After spray development of the PMMA with MIBK:IPA (methyl isobutyl ketone:isopropanol) and isotropic dry etching of the silicon with XeF 2 , the disks were located on silicon pedestals, with free-standing circumferences. The wafer was then thermally annealed at 125 °C on a hotplate, above the glass transition temperature of PMMA. Due to the along going reduction of the PMMA's surface free energy, the surface roughness of the disks is decreased and the disks form to the characteristic goblet shape. The fi nal edge-to-edge distance of the goblets was around 3 µm. Eventually, the wafers were split into chips comprising one laser pair each.
Refractive Index Measurements : The refractive indexes of the various solutions were measured by an Atago PAL-RI refractometer (Atago, Japan).