Bioconjugation techniques for microfluidic biosensors
- First Online:
- Cite this article as:
- Goddard, J.M. & Erickson, D. Anal Bioanal Chem (2009) 394: 469. doi:10.1007/s00216-009-2731-y
- 352 Views
We have evaluated five bioconjugation chemistries for immobilizing DNA onto silicon substrates for microfluidic biosensing applications. Conjugation by organosilanes is compared with linkage by carbonyldiimidazole (CDI) activation of silanol groups and utilization of dendrimers. Chemistries were compared in terms of immobilization and hybridization density, stability under microfluidic flow-induced shear stress, and stability after extended storage in aqueous solutions. Conjugation by dendrimer tether provided the greatest hybridization efficiency; however, conjugation by aminosilane treated with glutaraldehyde yielded the greatest immobilization and hybridization densities, as well as enhanced stability to both shear stress and extended storage in an aqueous environment. Direct linkage by CDI activation provided sufficient immobilization and hybridization density and represents a novel DNA bioconjugation strategy. Although these chemistries were evaluated for use in microfluidic biosensors, the results provide meaningful insight to a number of nanobiotechnology applications for which microfluidic devices require surface biofunctionalization, for example vascular prostheses and implanted devices.
KeywordsMicrofluidics/microfabricationBiosensorsDNA immobilizationSurface functionalizationBioconjugationSilane stability
The immobilization of nucleic acids onto glass and silicon substrates has relevance in a number of nanobiotechnology applications, including the detection of single nucleotide polymorphism [1, 2], disease surveillance for identification of emerging strains , and biosensor development [4–6]. In choosing a surface immobilization chemistry for these applications, there are a number of factors which must be considered. Of primary importance are probe density and uniformity; however, specific applications may have additional requirements. Biosensors that are intended to be reused multiple times and implanted biomedical devices must possess long-term stability in aqueous environments. Initial functionalization typically involves deposition of an organosilane monolayer; however, conjugation by a silane linkage may not provide the necessary long-term stability for these applications [7, 8]. In the case of many optical biosensors [9–11], it is important to limit the distance between the sensor surface and the immobilized probe in order to contain the hybridization event within the region of highest optical intensity. There are a few recent reports on conjugation of various ligands  and proteins  to silicon supports by 1,1-carbonyldiimidazole (CDI), indicating that it may be possible to directly link DNA capture probes to silicon substrates. Bypassing the need for a silane monolayer would bring the hybridization event closer to the substrate, a potential benefit to label-free detections.
Nucleic acid probe density is often reported in terms of the amount of immobilized capture probe; however, it has been reported that beyond an optimal probe density, steric hindrance can prevent hybridization efficiency [14, 15]. Therefore, while immobilized probe density is an important parameter to consider when selecting surface functionalization chemistries, it is important to also consider hybridization efficiency, which can be defined as the ratio of hybridized target nucleic acids to immobilized capture probes. Branched or dendritic tethers can increase immobilization density by providing more reactive sites per unit area for ligand immobilization. Tether molecules are also reported to improve hybridization efficiency in microarrays by reducing steric effects of the surface immobilized capture probe and bringing the hybridization event into a solution-like state [16–18]. Dendrimers in particular have been reported to improve binding capacity, homogeneity, stability, and hybridization efficiency in microarray applications [19–22].
Although there are many reports evaluating surface functionalization techniques for immobilizing nucleic acids on silica for microarray applications, there are few published data on the use of these techniques in microfluidic applications. Incorporating microfluidics in bioanalysis can reduce the necessary sample size, significantly reduce analysis time, and allow the potential for automated, miniaturized lab-on-a-chip biodevices [23–30]. However, the tight confinement of the flow layer in pressure-driven or electrokinetic microfluidic devices can exert high surface shear stresses, which may in turn affect the stability of immobilized biomolecules . The stability of surface functionalization chemistries and immobilized biomolecules to shear stresses exerted in microfluidic flow regimes has not been thoroughly investigated. It was therefore our goal to evaluate several DNA immobilization chemistries in order to compare immobilized capture probe density and hybridization efficiency in a microfluidic system. The bioconjugation chemistries evaluated herein are designed to perform on oxidized silica surfaces such as those presented on glass slides as well as the native oxide present on fabricated silicon devices. Each bioconjugation chemistry was evaluated for its stability under microfluidic flow and for long-term stability in an aqueous buffered environment. We compared two frequently used silicon surface functionalization techniques, glycidoxypropyltrimethoxysilane (GOPS) and aminopropyltrimethoxysilane (APTMS), as well as emerging techniques including direct linkage by CDI activation and linkage by a dendrimer tether. As a model system, we detected a synthetic oligonucleotide corresponding to human influenza A virus, subtype H3N2.
Superclean microarray slides were purchased from ArrayIt, TeleChem International (Sunnyvale, CA, USA). For contact angle and Fourier transform infrared (FTIR) measurements, chemistries were performed on P-type silicon wafers (WaferWorks, Helitek Company, Fremont, CA, USA). Glutaraldehyde (50%) and cyanoborohydride coupling buffer (3 g/L sodium cyanoborohydride) were purchased from Sigma Aldrich (St. Louis, MO, USA). Polydimethylsiloxane (PDMS, Sylgard 184) components were purchased from Dow Corning (Midland, MI, USA). 3-Glycidoxypropyltrimethoxysilane (GOPS, 97%) was purchased from Acros Organics (Morris Plains, NJ, USA). 1,1′-Carbonyldiimidazole (CDI) was purchased from Nova Biochem (Darmstadt, Germany). Generation 4.5 carboxylic-acid-terminated PAMAM dendrimers and Generation 5 amine-terminated PAMAM dendrimers were purchased from Dendritech (Midland, MI, USA). 3-Aminopropyltrimethoxysilane (APTMS), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), ficoll, formamide, N-hydroxysuccinimide (NHS), triethylamine (TEA), 2-(N-morpholino)ethanesulfonic acid (MES), sodium dodecyl sulfate (SDS), Triton X-100, 20× sodium saline citrate (SSC) buffer concentrate [20× SSC is 3 M NaCl, 0.3 M sodium citrate], and 10× phosphate-buffered saline (PBS) concentrate [1× PBS is 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer] were purchased from VWR (West Chester, PA, USA). All other reagents were laboratory grade or better and were used as received.
Sequences of DNA probes
Sequence (5′ to 3′)
[Amine]CAT GAT TGA AGC CGA GTC CTC[TAMRA]
[FL]GAG GAC TCG GCT TCA ATC ATG
To obtain optimal fluorescence signal, glass slides were used as substrates to quantify fluorescently tagged capture and target probes. To avoid the high background infrared absorption associated with glass substrates and thus enhance sensitivity, silicon wafers were used for grazing angle attenuated total reflectance FTIR (ATR-FTIR) analysis of the silane monolayers. The chemistries outlined below perform equally well on glass slides and the native oxide present on silicon wafers. Glass slides and silicon wafers were cleaned in piranha solution (3:1 mixture of concentrated sulfuric acid and 30% hydrogen peroxide) at 70 °C for 30 min, followed by rinsing in copious deionized water, in order to clean the surface and generate surface silanol (Si–OH) groups. Unless otherwise noted, surface-modified substrates were rinsed in deionized water, dried under 0.45 μm filtered air, and stored in clean glass petri dishes until further functionalization or analysis.
Functionalization of oxidized silicon substrates
Immobilization of DNA onto functionalized substrates
Although pH and use of specific bioconjugation reagents vary among the different immobilization chemistries, factors such as DNA concentration, salt concentration, ionic strength of buffer, reaction time, and spot size were kept constant in order to allow direct comparison of immobilization density between the various methods. Because the different functionalization procedures yielded different water contact angle (see “Results and discussion” below) spot size was maintained through the use of a PDMS mask (1.91-mm holes punched in a sheet of PDMS). A schematic of the use of PDMS to maintain DNA spot size is provided in the Electronic supplementary material. For each conjugation, 2.5 μl of DNA conjugation solution (detailed below) was spotted onto the substrate, confined within a PDMS well, and allowed to react at room temperature in the dark. To prevent drying during conjugation, substrates were stored over a moist filter round in a petri dish sealed with parafilm. After 7 h, DNA conjugation solutions were withdrawn from the PDMS wells, and the wells were rinsed twice with deionized water before being shaken for 5 min in rinse 1, rinse 2, rinse 3, and finally 1× PBS to remove non-covalently bound DNA. CDI—DNA capture probes were diluted to 10 μM in 0.15 M sodium carbonate buffer, pH 9.6, containing 1.0 M NaCl. DEND-GL—DNA capture probes were diluted to 10 μM in 0.15 M sodium carbonate buffer, pH 9.6, containing 10 mM sodium cyanoborohydride and 1.0 M NaCl. GOPS—DNA capture probes were diluted to 10 μM in 0.15 M sodium carbonate buffer, pH 9.6, containing 1.0 M NaCl. APTMS-GL—DNA capture probes were diluted to 10 μM in 0.15 M sodium carbonate buffer, pH 9.6, containing 10 mM sodium cyanoborohydride and 1.0 M NaCl. APTMS-DEND—Surface carboxylic acids were first activated in 50 mM MES, pH 6.0, containing 50 mM EDC, 5 mM NHS, and 0.5 M NaCl for 15 min at room temperature. Then, DNA capture probes were diluted to 10 μM in 0.15 M phosphate buffer, pH 7.4, containing 1.0 M NaCl.
Microfluidic hybridization assays
After functionalization of the substrates in bulk and immobilization of the DNA probes by confinement in PDMS wells, microfluidic hybridization assays were performed. Hybridization of synthetic target probes was conducted at room temperature under microfluidic flow, in a PDMS channel (1 mm × 2 cm × 45 μm) prepared by casting a 1:10 ratio of curing agent to elastomer base over a positive relief master . A schematic of the microfluidic channel is provided in the Electronic supplementary material. All reagents were withdrawn through the channel at 2 μl/min by a syringe pump. Hybridization buffer was drawn through the channel for 15 min to precondition the channel and substrate, after which target probes (diluted to 10 μM in hybridization buffer) were drawn through the channel for 15-min hybridization. After emptying the channel of hybridization mixture, rinse 1, rinse 2, and finally rinse 3 were withdrawn through the channel for 5 min each. PDMS fluidics were removed, the surface was air-dried, and fluorescence images were captured as described below.
Water contact angles of functionalized substrates (averages of n = 4 determinations) were determined using a VCA Optima contact angle analyzer (AST Products, Billerica, MA, USA). Contact angle was determined at each step in the surface functionalization prior to DNA immobilization.
FTIR spectroscopy was conducted on silane-treated substrates to ensure proper deposition of the APTMS and GOPS silane monolayers. FTIR spectroscopy was conducted on a Vertex 80v vacuum FTIR spectrometer using the VeeMAX™ II accessory equipped with a germanium ATR crystal and a zinc selenide polarizer set to parallel (p) polarization (Bruker Optik GmbH, Ettlingen, Germany). After taking a background spectra of piranha-cleaned silicon wafer, spectra of silicon wafers functionalized with APTMS or GOPS silane monolayers were collected using 256 scans with 4 cm−1 at 60° angle of incidence and were analyzed with KnowItAll® Informatics System 7.9 (Bio-Rad, Hercules, CA, USA).
Fluorescence images were taken of immobilized capture probes and hybridized target probes using a SensiCam CCD camera (Cooke Corporation, Eugene, OR, USA) through a ×4 objective. Image acquisition parameters were kept consistent in order to allow comparisons of fluorescence intensity between samples (250 ms, 1320 white point, 1520 black point for TAMRA modified capture probes; 250 ms, 500 white point, 100 black point for fluorescein modified target probes). Fluorescence intensities were determined using ImageJ Image Analysis program (National Institutes of Health, USA) and were converted to picomole per square centimeter through use of a standard curve made from dilutions of capture and target probe DNA in deionized water, spotted onto glass slides, dried, and imaged as described above. Initial immobilization density (number of immobilized capture probes per unit area before microfluidic flow), final immobilization density (number of immobilized capture probes per unit area after microfluidic flow), and hybridization density (number of hybridized target probes per unit area) were normalized by subtracting fluorescence of regions of functionalized substrates with no immobilized probes. Hybridization efficiencies were calculated as the ratio of hybridization density with respect to final immobilization density (after microfluidic flow).
Results and discussion
Contact angle of control and functionalized substrates
4.85 ± 0.30
9.33 ± 0.48
Amino-dendrimer immobilized via CDI
13.58 ± 0.93
Glutaraldehyde onto amino-dendrimer
22.75 ± 1.56
70.68 ± 0.28
43.78 ± 2.16
Glutaraldehyde onto APTMS
58.45 ± 0.86
Carboxy-dendrimer onto APTMS
13.68 ± 0.34
Variations in reaction conditions (time, concentration of reagents, etc.) make it difficult to draw comparisons between immobilization and hybridization densities observed in this work to other published reports; further, densities are often reported in terms of relative fluorescence units (RFU), which can only provide relative data. In an effort to quantify immobilization and hybridization densities, we have utilized a high-sensitivity, low-light camera to capture fluorescence images and compared mean RFU to a standard curve of synthetic DNA probe at varying concentrations at the same camera settings. The immobilization densities observed in this work are of the same order of magnitude as other reports, in which ssDNA has been immobilized in densities ranging from ~100 fmol/cm2 to ~20 pmol/cm2 [4, 5, 42, 43]. By directly observing the fluorescently tagged DNA immobilized on or hybridized to the substrate rather than performing a wash-off or cleavage assay, losses due to experimental error and photobleaching can be avoided.
Probe stability in aqueous environments
Stability of immobilized capture probes
Constant (A, pmol/cm2)
Decay rate constant (K, s−1)
Plateau (B, pmol/cm2)
Although APTMS monolayers have been reported to have low stability in aqueous environments [7, 8], use of a glutaraldehyde tether may have improved the APTMS stability by cross-linking the surface and by converting the reactive nucleophilic amines to secondary amine linkages, contributing to the relative stability of the APTMS-GL surface. GOPS functionalization had the second greatest stability, after APTMS-GL. As with APTMS-GL, the functionalized substrate is uncharged, and the surface is therefore less subject to microenvironmental pH extremes. As noted in the “Surface analysis” section (above), there may have been multilayer formation during GOPS deposition; therefore, the observed loss in stability may be a result of delamination of the multilayer. A major factor in the instability of immobilized probes for APTMS-DEND, DEND-GL, and CDI bioconjugations is likely the presence of surface microenvironmental effects. The numerous tertiary amines that make up the PAMAM dendrimer core are reported to be charged at neutral pH [37, 44, 45]. Similarly, piranha-cleaned glass used in the CDI linkage presents a charged surface, resulting in local pH extremes and increased rates of hydrolytic cleavage. Because conjugation with CDI involves a direct linkage of DNA to the glass substrate, its stability is highly dependent on nanoscale surface impurities. Although substrates were aggressively cleaned and were handled aseptically during treatment, deposition of an adventitious carbon layer may have also contributed to the poor stability.
Stability under microfluidic flow
Shear rate was determined to be 98.8 s−1, which when applied to the various functionalized surfaces for 45 min resulted in the following ranking of relative stabilities APTMS-GL (highest stability) > GOPS > DEND-GL > CDI > APTMS-DEND (lowest stability). This level of shear rate can also affect hybridization density and therefore efficiency. These values can be improved by modifying flow rate, channel dimensions, and hybridization buffer components. After 45-min microfluidic flow, CDI-functionalized substrates retained 32.0% of immobilized capture probe, while DEND-GL- and GOPS-functionalized substrates retained 74.5% and 77.8% immobilized capture probe, respectively. APTMS-GL-functionalized substrates had the greatest stability to shear stress, retaining 95.4% of the immobilized capture probe, while APTMS-DEND-functionalized substrates had the poorest stability to shear stress, retaining only 23.9% of the immobilized capture probe.
In general, the observed stabilities of the various bioconjugation chemistries to microfluidic flow conditions is similar to those observed after extended storage in aqueous environments, with the exception of DEND-GL functionalization. It is possible that glutaraldehyde cross-linked the amino-terminated dendrimer, providing enhanced stability to shear stress, whereas the major factor in the reduced stability of DEND-GL surfaces over extended periods is the formation of locally high pH from the charged dendrimer core, which is unaffected by glutaraldehyde cross-linking.
Immobilization density: APTMS-GL > GOPS > CDI > APTMS-DEND > DEND-GL
Hybridization density: APTMS-GL > CDI > APTMS-DEND > GOPS > DEND-GL
Hybridization efficiency: APTMS-DEND > CDI > DEND-GL > APTMS-GL > GOPS
Stability to shear stress: APTMS-GL > GOPS > APTMS-DEND > CDI > DEND-GL
Storage stability: APTMS-GL > GOPS > DEND-GL > CDI > APTMS-DEND
Although dendrimer tethers allowed for enhanced hybridization efficiency, as has been previously reported [16, 17], they did not increase density of immobilized capture probe, as was expected. Further, their poor stability to both microfluidic flow conditions and storage in buffered aqueous solution suggests that while they are advantageous in microarray applications, their suitability in microfluidic biosensors may be limited. Conjugation by APTMS-GL yielded the greatest immobilization and hybridization densities, as well as the greatest stability to both shear stress and extended storage in aqueous environment. Although it does not offer the stability of APTMS-GL-functionalized substrates, linkage of DNA capture probes to silicon substrates by CDI activation represents a promising bioconjugation technique in terms of density of immobilized probes and hybridization efficiency. CDI is a zero-length cross-linker, which activates carboxylic acids and hydroxyl groups to an intermediate that is reactive to nucleophiles, and considerably more stable than water soluble carbodiimide cross-linkers, thus allowing longer conjugation times in aqueous environments. Although it is widely used in peptide synthesis, biomolecule labeling, and immobilization of proteins onto organic and inorganic solid supports [41, 48–50], direct linkage of DNA to silicon substrates by CDI activation is an emerging bioconjugation technique. This chemistry could be particularly useful in the fields of label-free biosensing, in which highest device sensitivities are achieved when the biorecognition event occurs in close proximity to the sensing surface.
Important considerations in ensuring stability of silane monolayers includes proper cleaning of surface, formation of single monolayer, complete cross-linking of monolayer by curing after deposition, and minimizing hydrolysis of siloxane linkages by nucleophilic terminal groups. To that end, preventing the generation of GOPS multilayers through use of anhydrous conditions  may improve stability by avoiding delamination within the multilayer as was observed in this work. Further, glutaraldehyde provided improved stability both by further cross-linking the APTMS monolayer and by converting the nucleophilic primary amines to secondary amines, thus preventing nucleophilic attack on the siloxane linkage.
Although our focus was on isothermal microfluidic devices, future work evaluating thermal stability would provide useful additional information regarding suitability of these bioconjugation chemistries in thermocycling microfluidic applications such as polymerase chain reaction. Our primary interest regarding stability under shear stress is in the development of microfluidic biosensors; however, understanding the relative stability of these bioconjugation chemistries is important in a number of other nanobiotechnology applications, including development of vascular prostheses, drug delivery systems, and other implanted biomedical devices.
Support for this work was provided by the National Institutes of Health-National Institute of Biomedical Imaging and Bioengineering (NIH-NIBIB) under grant number R21EB007031. This work made use of STC shared experimental facilities supported by the National Science Foundation under Agreement No. ECS-9876771. The authors gratefully acknowledge Dr. Sam Nugen and Prof. Antje Baeumner for technical assistance in determining hybridization conditions and Sudeep Mandal for preparation of PDMS master.