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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
ACS Nano. Author manuscript; available in PMC May 24, 2012.
Published in final edited form as:
PMCID: PMC3178844

Optical Nanosensor Architecture for Cell Signaling Molecules Using DNA Aptamer-Coated Carbon Nanotubes


We report a novel optical biosensor platform using near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWNTs) functionalized with target-recognizing aptamer DNA for noninvasively detecting cell signaling molecules in real-time. Photoluminescence (PL) emission of aptamer-coated SWNTs is modulated upon selectively binding to target molecules, which is exploited to detect insulin using an insulin-binding aptamer (IBA) as a molecular recognition element. We find that nanotube PL quenches upon insulin recognition via a photoinduced charge transfer mechanism with a quenching rate of kq = 5.85×1014 M−1s−1 and a diffusion-reaction rate of kr = 0.129 s−1. Circular dichroism spectra reveal for the first time that IBA strands retain a four-stranded, parallel guanine quadruplex conformation on the nanotubes, ensuring target selectivity. We demonstrate that these IBA-functionalized SWNT sensors incorporated in a collagen extracellular matrix (ECM) can be regenerated by removing bound analytes through enzymatic proteolysis. As proof-of-concept, we show that the SWNT sensors embedded in the ECM promptly detect insulin secreted by cultured pancreatic INS-1 cells stimulated by glucose influx and report a gradient contour of insulin secretion profile. This novel design enables new types of label-free assays and non-invasive, in-situ, real-time detection schemes for cell signaling molecules.

Keywords: Aptamers, Biosensors, Carbon Nanotubes, Insulin, Near-infrared Fluorescence

Cell signaling molecules and cytokines secreted from cells carry information about cell functions and transmit it between cells. They are thus crucial components in cell-to-cell communication and immune responses, and measurement of these messenger molecules in-situ and in real-time without perturbing the cells is important for understanding and regulating cell functions and immune systems. Current methodologies of detecting such messenger molecules include immunoassays such as radio immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA),14 chromatographic methods,5, 6 and electrochemical measurements.79 Immunoassays have been widely used for quick screening and quantitative measurements using antigen-antibody reaction due to the simplicity, relatively low cost, and availability of reliable commercial kits. The chromatographic methods offer reliable analyte selectivity since the separation steps are included. The electrochemical measurements typically demonstrate high detection sensitivity and short signal response time for electrical signal readouts. However, these assays typically require harvest of the analyte molecules from the cellular environments and transfer to a predetermined testbed of the sensors. Thus, current detection technologies are not capable of noninvasively transmitting comprehensive, dynamic information about molecular targets in-situ and in real-time directly from the secreting cells.

Single-walled carbon nanotubes (SWNTs) show promise to meet these requirements as optical sensing materials for cell signaling molecules. Near-infrared (NIR) fluorescent properties render them excellent candidates for biological optical probes, since spectral information is transferred efficiently in the range of the so-called, tissue-transparent window, where absorption and autofluorescence by biological substances are minimal.1012 In contrast to commonly used organic fluorophores, carbon nanotubes are essentially non-photobleaching and non-blinking, which are advantageous in long-term measurements.13 SWNT photoluminescence (PL) is also very sensitive to molecular binding events at the sidewall, which may be exploited for optical detection schemes.1416 Additionally, the graphitic lattice allows non-covalent conjugation with biomolecular recognition elements such as DNA oligonucleotides, which preserves SWNT pristine electronic structures and thus their distinct optical signatures in aqueous solution.17, 18 Aptamers, single-stranded nucleic acids or peptides, are of great interest for this purpose, since they recognize specific molecular targets with high binding affinity, just like antibodies that recognize antigens. Aptamer oligonucleotides are advantageous as molecular recognition probes owing to their excellent stability, nontoxicity, reproducible synthesis, and ease of manipulation.19, 20 Aptameric sequences are typically identified by evolutionary in-vitro selection processes,21, 22 and several sequences have been shown to selectively bind cell signaling molecules by forming a unique secondary structure called the guanine (G) quadruplex.23, 24

In this work, we design and study novel optical nanosensor architecture based on NIR fluorescent SWNTs that non-invasively detect cell signaling molecules in real-time for the first time. Our new sensor platform includes SWNTs functionalized with aptamer DNA that modulate NIR emission upon recognizing signaling molecules secreted from live cells cultured on an extracellular matrix (ECM) in response to stimuli (Figure. 1). We use insulin and insulin-binding aptamer (IBA) as a model system of the analyte and the molecular recognition element in this study. Insulin, a polypeptide hormone from mammalian pancreatic cells, was chosen because of its implications in diabetes, as it controls the glucose levels in blood and insufficient release or loss of insulin results in the metabolic disease. A recently developed 30 base-long IBA sequence is used to selectively recognize insulin analytes.25 We have discovered for the first time that this aptamer retains a four-stranded, parallel G quadruplex conformation on SWNTs, a necessity for specifically binding to the molecular targets. We find that highly selective PL quenching upon analyte binding is caused by a photoinduced charge transfer mechanism, and a one-dimensional diffusion-reaction model is developed to describe target recognition processes. It should be noted that our approach is completely different from the molecular beacon type biosensing schemes where carbon nanotubes have been used as quenchers for organic fluorophores.26, 27 Finally, as proof-of-concept, we demonstrate that SWNT sensors embedded in a collagen ECM promptly detect insulin secreted by pancreatic INS-1 cells stimulated by glucose influx, demonstrating new types of label-free assays and in-situ, real-time detection schemes for cell signaling molecules.

Figure 1
Schematic of optical nanosensor architecture to measure insulin secreted from pancreatic INS-1 cells in-situ, in real-time. Near-IR fluorescent single-walled carbon nanotubes (SWNTs) are non-covalently functionalized with insulin-binding aptamer (IBA) ...


Design of Solution Phase Optical Biosensors

We first functionalized carbon nanotubes noncovalently with 30 base-long, insulin-recognizing DNA (i.e. insulin-binding aptamer or IBA) by two-stage dialysis against 1× Tris buffer. The dialysis processes replaced surfactants initially adsorbed on SWNTs with IBA strands via π-π interaction, and subsequently removed free oligonucleotides.17 Figure 2a shows the photoluminescence excitation (PLE) spectra of IBA-functionalized SWNTs (~11.2 mg/L), showing distinct NIR emission signatures of several SWNT species. A chiral index (n,m) that identifies individual nanotube species is assigned to each peak.28 Since CoMoCAT nanotubes that typically include a large population of small diameter (d ~ 1 nm) species are used in this study, strong fluorescence signals are observed from 950 to 1200 nm.29 Addition of insulin to the IBA-SWNT solution at 470 μM introduced significant emission quenching in all the observed nanotubes (Figure 2b). This signal transduction is consistent as there are no significant variations among different nanotube samples (Figure S1). The added insulin molecules diffuse in the solution and bind to IBAs on the nanotube sidewall, which results in SWNT signal transduction. The fluorescence spectra of SWNTs excited at 573 nm are shown as a function of insulin concentration in Figure 2c. The prominent (6,5) nanotubes emitting at ~995 nm and other species emission systematically quench with increasing insulin concentration from 0 to 470 μM. To determine the detection limit of IBA-functionalized SWNT sensors, the PL quenching of nanotubes is monitored at low insulin concentrations (Figure S2). The linear range of 0 to 180 nM insulin yields a detection limit of ~10 nM, which is higher than the insulin level in human blood, but sufficient to detect the insulin molecules in the human pancreas as the average pancreatic insulin concentration is approximately 10 μM.3032 In contrast to the drastic PL quenching, the optical absorption of IBA-coated SWNTs remains relatively constant in the presence of analytes (Figure 2d). This observation suggests that the mechanism of PL transduction is photoinduced excited-state electron transfer from the SWNT conduction band to the lowest unoccupied molecular orbital (LUMO) of the bound analyte.33, 34 This principle is consistent given that the reduction potential of insulin (red) is approximately −0.409 eV,35 and is positioned between the conduction and valence bands of SWNTs.36, 37 Figure 2e shows relative potential energies (black) of the valence and conduction bands of several nanotube species against normal hydrogen electrode (NHE) along with the optical absorption spectrum of IBA-SWNTs (blue).36, 37 The consistent quenching of all SWNT species observed in the PLE spectra also supports this mechanism (Figure 2a and 2b). The PL quenching kinetics can be determined from the Stern-Volmer formulation:


where I and Io are PL intensities with and without quenching molecules or Q. The slope of the linear relationship or Stern-Volmer constant is approximately KSV = kqτo = 1.17×104 M−1 (Figure 2f). The SWNT PL lifetime without a quencher (τo) is assumed to be 20 ps,38, 39 and thus the PL quenching rate, kq, is estimated to be roughly 5.85×1014 M−1s−1. These results strongly support photoinduced charge transfer as the predominant quenching mechanism.

Figure 2
NIR PL characteristics of IBA-coated SWNTs upon insulin recognition. (a) PLE spectra of IBA-SWNTs in 1× Tris buffer, showing distinct NIR emission signatures of several SWNT species. (b) PLE profile in the presence of insulin at 470 μM, ...

Of considerable utility is that IBA-coated SWNTs do not optically respond to other interfering proteins (Figure 3a). To verify the target selectivity of the SWNT sensors, we examined control proteins with a wide range of isoelectric points (pI). Bovine serum albumin (BSA) and proteinase K have pI 4.8 and 8.9, which are correspondingly negatively and positively charged under our buffer conditions at pH 8.0. Both control proteins do not modulate the SWNT emission significantly, whereas 82% PL quenching is observed with insulin of the same concentration, demonstrating the analyte selectivity of IBA-SWNTs. This selectivity is highly dependent on the specific DNA aptamer sequence used as the molecular recognition element in the sensor platform. We note that the IBA forms a G-quadruplex conformation in order to selectively bind to target insulin.25 We used circular dichroism (CD) spectroscopy to probe the secondary structure of the sequence, and the CD spectrum of IBA bound to SWNTs is compared to that of free strands in solution (Figure 3b). Both spectra show two positive peaks at approximately 263 and 209 nm, and a negative valley at ~240 nm, which indicates a four-stranded, parallel G-quadruplex conformation.4042 Thus, these CD spectra suggest that the IBA strands retain the four-stranded, parallel G-quadruplex structure on and off the nanotubes. This observation is the first to monitor DNA G-quadruplex formation at the nanotube sidewall, rendering the SWNT sensors highly selective toward target insulin. The CD results imply that the optical biosensor architecture based on aptamer-coated SWNTs may be applicable to other sequences that require G-quadruplex formation for specific target recognition.

Figure 3
Selectivity of IBA-coated SWNTs for insulin recognition. (a) SWNT PL intensities in the presence of control proteins with various isoelectric points (470 μM). The control proteins do not significantly modulate SWNT PL, whereas insulin immediately ...

Kinetic Model of Analyte Recognition Using SWNT PL Quenching

To understand the kinetics of selective analyte binding that modulate SWNT emission, we develop a simple one-dimensional kinetic model based on molecular diffusion and reaction, while considering insulin diffusion and binding with IBA-SWNTs in solution.43 We consider that the analyte molecules are transported by diffusion, while the fluorescent sensors are homogeneously distributed in the domain as shown in Fig. 4a. The diffused analyte molecules are assumed to bind to the sensors irreversibly, resulting in PL signal transduction. The reaction between the analyte and sensor is expressed as:


where kr denotes the reaction rate. Initially, the analyte molecules (Uo) and sensor molecules (Vo) are uniformly situated in the space interval [0, z] and [0, L], respectively. Here, Uo and Vo denote the initial concentrations of analytes and sensors. The rate equation of the diffusion-reaction model is:


Boundary condition:xu(0,t)=xu(L,t)=0fort>0

Initial condition:u(x,0)={U0if0<x<z0otherwise}

where u(x,t) is the sensor concentration and D is the diffusivity of analyte molecules in solution. Using separation of variables and Fourier series, an analytic solution is obtained:



Considering the variation of the analyte concentration at a position x at time t, the bound analytes and sensors in the time interval [0, t] are:


where A is the molar ratio between analyte molecules and sensors. We assume that the analytes do not fluoresce and that the PL intensity of the sensors decreases with a quenching constant,θ, when the sensor recognizes the analyte. Then, the normalized PL signal can be expressed as:


We consider the detection region of [x−r, x+r], and the final emission signal becomes:


We used this simple one-dimensional diffusion-reaction model to determine the reaction rate between insulin and IBA-SWNTs in solution. To experimentally determine the kinetics of insulin recognition by IBA-SWNTs, we measured nanotube emission over 150 seconds after insulin injection (Fig. 4b). In our experiments, z and L were approximately 377 μm and 1.39 mm, while the focal length of the objective lens was h = 190 μm and the detection region or the field of depth was approximately 2r = 0.4 μm. The diffusivity of insulin is estimated at D = 150 μm2/s.44 As shown in Fig. 4b, the kinetic model describes the experimental data well and suggests a diffusion-reaction rate of kr = 0.129 s−1, which is similar to that of SWNT reaction with biomolecules reported by Satishkumar et al.33

Figure 4
Kinetics of SWNT PL signal changes upon recognition of the analyte molecules. (a) Domain for a one-dimensional kinetic diffusion-reaction model. In this model, the analyte molecules are initially located in 0 < x < z and diffuse into the ...

Integration of SWNT Sensors into an ECM

To interface nanotubes with cellular environments, we first incorporated IBA-coated SWNTs into a biocompatible collagen ECM that can be used as a cell culture platform since it facilitates the growth and differentiation of attached cells.45, 46 The collagen ECM immobilizes IBA-SWNTs inside its porous structure and prevents them from directly contacting cultured living cells. The immobilized nanotubes appear to be randomly spread in the matrix as shown in the NIR PL images (Figure 5a and 5b), where a 658 nm laser was used for excitation and SWNT emission longer than 850 nm was collected under an inverted microscope. This excitation energy produces the most prominent (7,5) nanotube emission at ~1044 nm among several SWNT species (Figures S3 and and5c5c).29 The emission characteristics of SWNTs immobilized in the ECM remain almost constant; the PL wavelength of each species corresponds to that of nanotubes uniformly dispersed in solution (Figure S3). After addition of insulin at 470 μM, a drastic SWNT PL quenching is observed in both the NIR images (Figure. 5b) and the corresponding spectra (Figure. S4), similar to what is seen in solution phase. The porosity of the collagen ECM structure allows small molecules such as insulin (5.8 kDa) to diffuse into the matrix and react with immobilized IBA-SWNTs, resulting in emission transduction.47

Figure 5
Reversible fluorescence of IBA-SWNTs immobilized in a collagen ECM. NIR PL images of IBA-SWNTs embedded in an ECM (a) before and (b) after insulin injection at 470 μM. (c) The corresponding spectra of NIR images (a) and (b) are shown in blue and ...

The quenched SWNT fluorescence is restored by incorporating α-chymotrypsin, an enzyme that digests insulin molecules.9, 48 After added to the solution at 49 μM, α-chymotrypsin (25 kDa) diffuses into the ECM and hydrolyzes the insulin bound to nanotube surface, resulting in full recovery of SWNT-PL (from red to green in Figure 5c). This proteolysis process typically takes approximately 1 hour. The IBA-SWNTs in the ECM are reusable after washing with excess buffer to remove the enzyme and insulin fragments. Thus, subsequent addition of insulin to the regenerated nanotube sensors reinduces the PL modulation, which can then be recovered with α-chymotrypsin. Figure 5d shows two cycles of insulin binding and removal by proteolysis. The recycled SWNT sensors were examined two months later and their optical signals were persistent, which may be readily used for target recognition again. In theory, these sensors may be regenerated for an indefinite number of cycles. Given the SWNTs' photostable optical properties without photobleaching, the IBA-SNWT sensors are capable of a large number of uses and long-term operation and have a potential for reusable sensors in the cellular environment.

Insulin Detection Secreted from Pancreatic INS-1

As proof-of-concept, we demonstrated in-situ, non-invasive, real-time detection of insulin secreted from pancreatic INS-1 cells. Approximately 7.5×105 cells were plated and cultured on a collagen ECM that contained the immobilized IBA-SWNTs. After incubation for 24 hours at 37°C, the cells were visualized using differential interference contrast (DIC) microscopy (Figure 6a), while NIR PL images of SWNTs were recorded every second for 5 minutes after each glucose injection. During this period, high doses of glucose were added three times to facilitate insulin secretion from the cells.49, 50 Figure 6b and 6c shows the NIR PL images before and 15 minutes after glucose addition. The PL quenching map overlaid with the DIC cell image (Figure 6d) is constructed by subtracting the PL intensity of each pixel in Figure 6c from that in Figure 6b, so white indicates maximum PL transduction of IBA-SWNTs under our experimental conditions. This pseudo-colored map represents the spatially resolved insulin secretion profile around the observed cell; a higher degree of nanotube quenching is observed near the cell, and the quenching level decreases away from the cell. The added glucose facilitates cellular secretion of insulin molecules which are transported away from the cell by diffusion, which is shown in the plotted gradient contour of insulin outflux. The PL intensity at two positions in Figure 6d is plotted as a function of time in Figure 6e. The time-resolved insulin profiles at these locations show that each glucose addition event promotes metabolic activities in the cells, which in turn results in secretion and diffusion of insulin molecules. The SWNT sensors at location #1 that is beneath the observed cell demonstrate greater signal transduction compared to those at location #2 away from the cell, indicating outflux of insulin molecules. We confirm that this PL transduction is not caused by any molecules other than analyte insulin. As shown in Figure 6f, cell culture media (RPMI 1640) or high doses of glucose (0.3 M) do not result in any apparent changes in nanotube emission signals.

Figure 6
In-situ, real-time detection of insulin secreted from cultured pancreatic INS-1 cells on a collagen ECM containing NIR fluorescent IBA-SWNTs. (a) Differential interference contrast (DIC) image of a single INS-1 cell. NIR PL images of IBA-SWNTs in the ...


In summary, we have shown that DNA aptamer-coated SWNTs can form the basis for label-free optical nanosensors to remotely monitor cell signaling molecules in real-time. The carbon nanotubes integrated with an insulin-recognizing DNA sequence demonstrate strong PL transductions upon insulin binding via a photoinduced charge transfer mechanism. Quantitative measurements of SWNT PL quenching suggest a detection limit of ~10 nM for insulin. The CD measurement reveals that a four-stranded, parallel G-quadruplex structure of the aptamer DNA is retained on the SWNT, allowing the high target selectivity. The Stern-Volmer plot yields a PL quenching rate of approximately kq = 5.85×1014 M−1s−1, while one-dimensional kinetic model for analyte recognition suggests a diffusion-reaction rate of kr = 0.129 s−1. We have demonstrated that the SWNT sensors embedded in a biocompatible collagen ECM can be regenerated by enzymatic proteolysis using α-chymotrypsin. The optical sensors are capable of reporting a gradient contour of insulin outflux from cultured pancreatic INS-1 cells stimulated by glucose, demonstrating new types of in-situ, real-time detection schemes for cell signaling molecules. The biosensor architecture developed in this study may be applied to other analytes of interest by incorporating appropriate aptamer sequences, and could improve the detection sensitivity to the single analyte molecule level if individual nanotubes are examined.5153



Insulin-binding aptamer (IBA: 5′-GGT GGT GGG GGG GGT TGG TAG GGT GTC TTC-3′) strands were custom-synthesized and purchased from Integrated DNA Technologies, Inc. Several DNA sequences have been reported to selectively bind to insulin molecules. For example, the insulin-linked polymorphic region sequence (ILPR2: 5′-ACA GGG GTG TGG GGA CAG GGG TGT GGG G-3′) can capture insulin molecules.24 Because the IBA sequence reportedly has a higher binding affinity to insulin than ILPR2,25 the IBA sequence was selected as molecular recognition elements for DNA-coated SWNT sensors in this study.

CoMoCAT SWNTs were purchased from Southwest Nanotechnology, while sodium cholate was obtained from Affymetrix Inc. Insulin from bovine pancreas, BSA, Proteinase K, β-mercaptoethanol, HEPES buffer, Na-pyruvate, L-glutamine, penicillin, and streptomycin were purchased from Sigma Aldrich. We obtained fetal calf serum, trypsin EDTA solution, RPMI 1640 medium, and phosphate-buffered saline (PBS) from Invitrogen, while rat tail collagen I was purchased from BD Bioscience.

SWNT suspension

Aqueous suspensions of sodium cholate (SC)-coated SWNTs (~181.10 mg/L) were prepared by tip sonication at 20 W for 1 hour, followed by ultracentrifugation at 30,000 rpm.29 After centrifugation, the supernatant was carefully decanted to obtain homogeneous surfactant-suspended SWNT samples which were separated from the denser catalyst particles, bundles, and impurities. In order to non-covalently functionalize nanotubes with DNA, approximately 91 μM of IBAs were added to the SC-SWNT solution which were dialyzed using a 12–14 kDa porous membrane (Fisherbrand) against 1× Tris buffer for 24 hours.16 The buffer was changed every 4 hours during the dialysis, and aqueous SWNT solutions were maintained at pH 8.0 using NaOH solution. To remove free DNA from the SWNT suspension, a second dialysis using a 100 kDa membrane (Fisherbrand) was performed for 24 hours.

Integration of IBA-SWNTs in the ECM

Collagen ECM was prepared following the procedure described by Han et al.47 Approximately, 93 μL of 0.1 M NaOH solution was added to 410 μL rat tail collagen I (3–4 mg/mL) to achieve pH 7.5. Then, 20 mL 1× PBS buffer and 5 mL IBA-coated SWNTs were added to pH-controlled collagen solution and stirred for 5 minutes in an ice bath to maintain collagen stability. A 100 μL aliquot of collagen solution containing IBA-SWNTs was then pipetted onto a glass bottom petri dish (MatTek Corp.) and polymerized for 1 hour at 40°C. During polymerization, parafilm was wrapped over the petri dish to prevent the solution from evaporating.

Cellular insulin detection

Pancreatic INS-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 μM β-mercaptoethanol, 10 mM HEPES, 1mM Na-pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg streptomycin on 10 cm BD Falcon polystyrene tissue culture dishes at 37°C and a 5% CO2 atmosphere. Once the cells reached 90% confluency, they were detached from the culture dish using a 0.05% trypsin EDTA solution. The cells were then centrifuged and resuspended in 4 mL of growth medium. Prior to plating, the IBA-SWNT containing collagen ECM was sterilized with UV light for 20 minutes. Suspended cells (~750 cells/μL) were then pipetted onto three spots (~0.3 mL per spot) on the sterilized collagen ECM and incubated for 24 hours in 3 mL RPMI 1640 medium.

Optical measurements

NIR fluorescence spectra of IBA-coated SWNTs in solution were measured using a Horiba Jobin Yvon Fluorolog-3 spectrofluorometer with a liquid N2-cooled InGaAs detector. The integration time for measuring a single point was 10 seconds and the slit widths of excitation and emission are 5 nm and 10 nm, respectively. A home-built microscope system measured NIR fluorescence images and spectra of IBA-SWNTs in the collagen ECM using an OMA-V 2-D liquid N2-cooled InGaAs camera (320×256 pixels, Princeton Instruments) with a 658 nm diode laser excitation. A Zeiss Plan-Apochromat 63×/1.4 oil-immersion objective lens was used to collect the nanotube emission. Optical absorption spectra were measured with a Perkin Elmer Lambda 950 UV/VIS/NIR spectrophotometer. CD spectra of free IBA strands and IBA-SWNTs in solution were measured with a JASCO J-810 spectropolarimeter using a 0.1-cm path-length quartz cuvette at room temperature.

Supplementary Material



This work was supported by National Science Foundation and Purdue University. J.H.C. acknowledges a NSF CAREER Award and B.A.B. is grateful for a NSF Graduate Research Fellowship. The INS-1 cells were provided as a kind gift by Dr. Raghu Mirmira of Indiana University.


BRIEFS: Near-IR fluorescent single-walled carbon nanotubes functionalized with target-recognizing aptamer DNA noninvasively detect signaling molecules secreted from live cells in real-time via photoinduced charge transfer mechanism.

Supporting Information Available: The determination of the detection limit of IBA-SWNT sensors; spectra of IBA-SWNTs in buffer solution and in an ECM; regeneration of SWNT sensors in solution; polarization of IBA-SWNTs in the collagen ECM; This material is available free of charge via the Internet at http://pubs.acs.org.


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