Ultrasensitive microchip based on smart microgel for real-time online detection of trace threat analytes

Significance

Real-time detection of trace threat analytes is critical for global sustainability. The key challenge is how to efficiently convert and amplify the analyte signal into simple readouts. Here we report an ultrasensitive microfluidic platform incorporated with stimuli-responsive smart microgel for real-time detection of trace threat analytes. The microgel swells in response to specific analyte, thus converting trace analyte concentration into significantly amplified signal of flow rate change for highly sensitive, fast, and selective detection, which can be monitored on cell phone for timely warning and terminating of pollution. This work provides a generalizable platform for incorporating myriad microgels to achieve ever-better performance for real-time detection of various trace threat molecules, and may expand the scope of applications of detection techniques.

Abstract

Real-time online detection of trace threat analytes is critical for global sustainability, whereas the key challenge is how to efficiently convert and amplify analyte signals into simple readouts. Here we report an ultrasensitive microfluidic platform incorporated with smart microgel for real-time online detection of trace threat analytes. The microgel can swell responding to specific stimulus in flowing solution, resulting in efficient conversion of the stimulus signal into significantly amplified signal of flow-rate change; thus highly sensitive, fast, and selective detection can be achieved. We demonstrate this by incorporating ion-recognizable microgel for detecting trace Pb²⁺, and connecting our platform with pipelines of tap water and wastewater for real-time online Pb²⁺ detection to achieve timely pollution warning and terminating. This work provides a generalizable platform for incorporating myriad stimuli-responsive microgels to achieve ever-better performance for real-time online detection of various trace threat molecules, and may expand the scope of applications of detection techniques.

Timely detection of trace threat analytes that are harmful to environment and human health is critical for environmental protection (1), disease treatment (2, 3), and epidemic prevention (4). The key challenge is how to efficiently convert and amplify the analyte signal into simple readouts for real-time detection. Based on the stimuli-responsive volume changes of smart hydrogels (5, 6), current techniques allow converting the stimulus signals into electrical or optical signals for detection. Generally, the volume change of smart hydrogels can be converted to electric current through field-effect transistors and pressure sensors for detecting glucose (2.8 × 10⁻³ M) (7) and metal ions (8). These methods inefficiently use the 3D hydrogel deformation, thus possessing poor detection limit. Improved sensitivity can be achieved by converting the target signals into optical signals. Typically, upon volume change, photonic crystal hydrogels can change their lattice constants to shift diffraction peaks for detecting trace analytes such as Pb²⁺ (∼10⁻⁹ M) (9, 10), DNA (10⁻⁹ M) (11), and 3-pyridinecarboxamid (12) via spectrometer, whereas hydrogel diffraction gratings can adjust their refractive indexes to change the diffraction efficiency for detecting Ig-G (∼6 × 10⁻⁶ M) (13) and glucose (∼2.3 × 10⁻⁵ M) (14) via silicon photodiodes, resistors, and preamp module. Alternatively, in response to target signals, hydrogel microcantilever can slightly bend to deflect laser light for detecting Pb²⁺ (10⁻⁷ M) (15) and CrO₄²⁻ (10⁻¹⁰ M) (16) via atomic force microscopy, whereas fluorescently modified hydrogels can change their fluorescent intensity for detecting glucose (17), Hg²⁺ (10⁻⁸ M), and Pb²⁺ (10⁻⁹ M) (18) via fluorescence spectrophotometers. However, all these techniques require sophisticated equipment and professionals for detection and analysis. To sum up, the platforms with electrical signals provide easy use and low cost, but the detection limit is poor, whereas the platforms with optical signals offer improved sensitivity, but require sophisticated analyzing protocols, which restrict the applications for real-time online detection. Up to now, development of simple and ultrasensitive detection platforms for real-time online detection of trace analytes has remained a challenge.

Herein, we report an ultrasensitive microchip based on stimuli-responsive smart microgel for real-time detection of trace threat analytes. The key unit of our detection platform is a microfluidic chip with glass-capillary microchannel integrated with cylinder-shaped smart microgel, which allows highly sensitive, fast, and selective detection of trace threat analytes. We demonstrate this by using poly(N-isopropylacrylamide-co-benzo-18-crown-6-acrylamide) [P(NIPAM-co-B18C6Am)] microgel with NIPAM units as actuators and B18C6Am units as ion signal sensing receptors to selectively recognize trace Pb²⁺ (Fig. 1). As illustrated in Fig. 1A, the microgel is initially shrinking in water at operation temperature (T_o) (Fig. 1B), which is above the volume phase transition temperature (VPTT) of the microgel in pure water (VPTT₁). When trace Pb²⁺ appears, the B18C6Am units capture Pb²⁺ and form stable B18C6Am/Pb²⁺ host–guest complexes via molecular recognition (6, 19) (Fig. 1 B and C). This leads to a VPTT shift from VPTT₁ to a higher VPTT₂ due to the electrostatic repulsion among the charged B18C6Am/Pb²⁺ complex groups (19–21). Thus, the microgel isothermally swells at T_o, due to the shift of VPTT to a higher value than T_o and the enhanced osmotic pressure within the microgel based on Donnan potential (19–21). By fabricating cylinder-shaped P(NIPAM-co-B18C6Am) microgel inside a capillary microchannel, the interstice between the microgel and capillary creates a crescent-moon-shaped microspace for flowing fluids (Fig. 1 D and E). Upon recognizing Pb²⁺, the microgel isothermally swells to a certain degree depending on the Pb²⁺ concentration ([Pb²⁺]) (19, 21); as a result, the flowing area of the crescent-moon-shaped microspace decreases, and thus the flow rate drops correspondingly (Q → Q′, in which Q > Q′). According to the Hagen–Poiseuille law, the flow rate through a microchannel is governed by the fourth power of the hydraulic equivalent diameter of flowing space (22). Thus, the Pb²⁺-induced swelling of the P(NIPAM-co-B18C6Am) microgel in capillary microchannel greatly affects the flow rate. Therefore, with the proposed microchip, the trace Pb²⁺ signals can be efficiently converted into significantly amplified signals of flow rate change. Then, measurement of flow rates downstream the microchip via a simple online flowmeter (Fig. 1 F–H) allows quantitative detection of trace Pb²⁺. Because the characteristic time of gel swelling is proportional to the square of a linear dimension of the hydrogel (23), the micrometer-scale size of microgel enables its rapid swelling upon recognizing Pb²⁺. Furthermore, the flowing of solution around the microgel enables enhanced Pb²⁺ transfer into the microgel networks, which is also beneficial to the rapid swelling of microgel. As a result, ultrasensitive and real-time detection of Pb²⁺ can be achieved with the proposed platform.

Fig. 1.

Schematics of Pb²⁺-detection platform equipped with microchip incorporating P(NIPAM-co-B18C6Am) microgel. (A–C) P(NIPAM-co-B18C6Am) hydrogel can isothermally swell after recognizing Pb²⁺ via forming stable host–guest complexes. (D and E) By incorporating cylinder-shaped P(NIPAM-co-B18C6Am) microgel inside the capillary as Pb²⁺ sensor, the trace Pb²⁺ signal can be efficiently converted into significantly amplified signal of flow rate change (Q → Q′, in which Q > Q′). (F–H) Pb²⁺-detection platform (F), equipped with a microfluidic chip for Pb²⁺ sensing based on the above-mentioned principle and an online flowmeter for flow-rate measuring based on the flow-rate–dependent temperature distribution (G and H), enables real-time online quantitative detection of trace Pb²⁺.

Results and Discussion

Fabrication of Microfluidic Chip Integrated with Cylinder-Shaped Microgel.

To fabricate the microfluidic chip integrated with cylinder-shaped microgel, an advanced rotation-based method is developed for 360° uniform UV irradiation of the mask-covered capillary (Fig. 2 A and B), which is filled with aqueous solution containing monomers B18C6Am and NIPAM, cross-linker N,N′-methylene-bis-acrylamide, and photoinitiator 2,2′-azobis(2-amidi-nopropane dihydrochloride). This approach efficiently converts the loaded monomer solution (Fig. 2 C1 and D1) into uniform cylinder-shaped microgel (Fig. 2 C2 and D2, SI Appendix, Fig. S1, and Movie S1). Then, a coaxially placed cylinder bar of stainless steel is skillfully introduced to stably support the microgel inside the capillary microchannel (Fig. 2 C3 and D3 and SI Appendix, Fig. S2). After removing the unpolymerized solution from the microchannel (Fig. 2 C4 and D4), microgel-based Pb²⁺ sensor is obtained. Finally, the Pb²⁺ sensor is fixed on a glass plate and simply connected to an online flowmeter by polyethylene tubes for constructing the Pb²⁺-detection platform (SI Appendix, Fig. S3). The relationship between the swelling rate and microgel size has been investigated by transferring the microgel from pure water to 10⁻⁵ M Pb²⁺ solution. The ratio of the microgel volume at time t s to that at 0 s after the transfer (N_V) decreases with increasing the microgel radius at a fixed time, indicating a faster swelling rate of smaller microgel (Fig. 2E). The experimental data of the characteristic time for hydrogel swelling (τ_v), defined as the time at which the hydrogel reaches 99% of its equilibrated swelling volume, coincide well with the calculated values obtained by the equation τ_v = r²/(π² × D) (23) (Fig. 2F), in which D is the collective diffusion coefficient determined by the kinetic experiment, and r is the microgel radius.

Fig. 2.

Fabrication of microgel-based Pb²⁺ sensor. (A and B) An advanced rotation-based method is developed for fabricating uniform cylinder-shaped microgel in capillary microchannel, by inserting monomer-solution–containing capillary fixed on a motor into two steel tubes (A) that are covered by a mask to form an exposed part between tubes, and then using rotation-assisted 360° uniform UV irradiation to achieve polymerization (B). (C and D) Schematics (C) and optical micrographs (D) showing the fabrication and fixation of microgel. In the monomer-solution–containing microchannel (C1 and D1), uniform cylinder-shaped microgel is synthesized by rotation-assisted UV-polymerization (C2 and D2), and then stably supported by skillfully introducing a coaxially placed stainless steel bar (C3 and D3), followed with removing the unpolymerized solution (C4 and D4). (E) Effect of the microgel radius on the dynamic swelling ratio (N_V = V_t/V₀) of the cylinder-shaped microgel, where V₀ and V_t are, respectively, the microgel volume at 0 s and t s after transferring from pure water to 10⁻⁵ M Pb²⁺ solution. (F) Characteristic time for the swelling of microgels (τ_v) with different radius.

Sensitivity of Detection Platform.

Our detection platform exhibits highly sensitive and fast detection performance. First, we determine that the optimal operation temperature of our platform is 34 °C, because the sensor shows the most significant equilibrated change of flow rate (ΔJ = Q – Q′) in response to each [Pb²⁺] at 34 °C compared with the flow rate of pure water (Q) (Fig. 3A). It is worth noting that, at 30 °C, the equilibrated swelling size of microgel is still larger than the microchannel dimension; thus, the microgel volume is constrained by the capillary wall for blocking the microchannel. So, there is no flow in the microchannel and the ΔJ remains nearly unchanged at 30 °C. At 34 °C, significant decrease of flow rate can be detected after recognizing Pb²⁺ with [Pb²⁺] varied from 10⁻¹⁰ M to 10⁻⁵ M within 5 min (Fig. 3B) (see SI Appendix, Fig. S4 for detailed flow rate data within the first 250 s). Especially, even in the [Pb²⁺] range from 10⁻¹⁰ M to 10⁻⁸ M, which is much lower than the guideline value of the World Health Organization for drinking water (4.83 × 10⁻⁸ M), obvious decrease in flow rate can be detected, indicating the ultrasensitivity of our platform. Meanwhile, the slope of ΔJ_t/ΔJ_max curves (more details see SI Appendix, Fig. S5) at ΔJ_t/ΔJ_max = 50% (S₅₀) increases with increasing [Pb²⁺], where ΔJ_t is the ΔJ at time t s and ΔJ_max is the maximum value of ΔJ (Fig. 3C). Moreover, the times required for change of ΔJ_t/ΔJ_max from 5% to 50% (t₅₀–t₅) and for change of ΔJ_t/ΔJ_max from 5% to 90% (t₉₀–t₅) both decrease with increasing [Pb²⁺] (Fig. 3C, Inset). All of the results indicate a faster dynamic swelling rate at higher [Pb²⁺] due to the faster formation of B18C6Am/Pb²⁺ complex groups. For accurate detection of [Pb²⁺], the quantitative relationship between Pb²⁺ concentration and flow rate change is obtained from Fig. 3D, as [Pb²⁺]=3 × 10⁻¹⁴ × (ΔJ)^4.3.

Fig. 3.

Highly sensitive and fast detection of Pb²⁺. (A) Effect of temperature and [Pb²⁺] on the equilibrated change of flow rate (ΔJ = Q – Q′) after switching pure water to Pb²⁺ solution for 15 min. (B) Time-dependent flow-rate changes in response to different [Pb²⁺] values at 34 °C. (C) Effect of [Pb²⁺] on the slope (S₅₀) of ΔJ_t/ΔJ_max curves at ΔJ_t/ΔJ_max = 50% in SI Appendix, Fig. S5. (Inset) Effect of [Pb²⁺] on the time required for change of ΔJ_t/ΔJ_max from 5% to 50% (t₅₀–t₅) and for change of ΔJ_t/ΔJ_max from 5% to 90% (t₉₀–t₅). (D) Quantitative relationship between [Pb²⁺] and ΔJ after switching pure water to Pb²⁺ solution for 15 min.

Selectivity and Repeatability of Detection Platform.

Our detection platform also shows excellent selectivity and repeatability for Pb²⁺ detection. Interferences from other ions in flow rate decrease only occur when the ion concentrations are larger than 10⁻⁶ M (Fig. 4A). Under concentration of 10⁻⁶ M, Pb²⁺ results in a significant decrease of flow rate (60 μL/min), while interferences from other ions are all negligible (less than 2 μL/min) (Fig. 4B). Such interferences from Ba²⁺, Sr²⁺, K⁺, and Na⁺ are negligible even when increasing their concentrations 100× that of [Pb²⁺] at [Pb²⁺] = 10⁻⁶ M, or even 1,000× that of [Pb²⁺] at [Pb²⁺] = 10⁻⁵ M (Fig. 4C), indicating excellent selectivity. Moreover, the detection platform can be repeatedly used by simply and alternatively washing off the captured Pb²⁺ with water at 55 °C and 25 °C (SI Appendix, Fig. S6). In water at 55 °C (>VPTT₂), the microgel shrinks and makes the B18C6Am units close to each other, producing electrostatic repulsions among the ions against the formation of stable B18C6Am/Pb²⁺ complexes, and leading to decomplexation of Pb²⁺ from B18C6Am units (20). Meanwhile, the decrease of inclusion constant upon heating also facilitates the Pb²⁺ decomplexation (21). In water at 25 °C, the microgel swells again and takes fresh water inside for Pb²⁺ removal. Repeat of such a shrinking/swelling cycle upon heating and cooling enhances the water transporting into and out of the microgel network for removing Pb²⁺. The detection platform after different wash cycles is used for Pb²⁺ detection to estimate the recovery of the detecting performance. For detection platforms used for detecting different [Pb²⁺], each of the flux recovery ratios [R_F = (ΔJ_max – ΔJ_t)/ΔJ_max] can reach 100% after different wash cycles (Fig. 4D). The cycle times required for 100% recovery increase with increasing [Pb²⁺] (Fig. 4D and SI Appendix, Fig. S7A). The detection platforms show excellent recovery performance for repeated detection with high accuracy (SI Appendix, Fig. S7B). The detection mechanism and portability of our detection platform enable its flexible and facile utilization as an online unit for real-time detection of Pb²⁺. This is demonstrated by using the platform for real-time online detection of Pb²⁺ in tap water and in wastewater from a model industrial factory for pollution warning and terminating via cell-phone monitoring (SI Appendix, Figs. S8–S10 and Movies S2 and S3).

Fig. 4.

Highly selective and excellent repeatability of Pb²⁺-detection platform. (A) Equilibrated flow rates at different concentrations of Na⁺, Sr²⁺, K²⁺, Ba²⁺, and Pb²⁺ at 34 °C. (B) Effect of ion species on the equilibrated change of flow rate ΔJ after switching pure water to the solution containing each ion (10⁻⁶ M) for 15 min at 34 °C. (C) Effects of interfering ions on the flow rate changes (ΔJ). The interfering ions include Ba²⁺, Sr²⁺, K⁺, and Na⁺. (D) Dynamic flux recovery ratio (R_F) of detection platforms after detecting different [Pb²⁺] concentrations and washing with pure water in a wash cycle manner shown in SI Appendix, Fig. S6.

Conclusions

We have demonstrated the real-time detection of Pb²⁺ by developing ultrasensitive microchips incorporating P(NIPAM-co-B18C6Am) smart microgel. The proposed detection platform exhibits highly sensitive, fast, and selective detection performance, and possesses flexible and facile utility as an online unit for real-time Pb²⁺-detection. Especially, the measured [Pb²⁺] value can be conveniently displayed on the popular cell phone, with which timely warning or even terminating of Pb²⁺ pollution can be easily achieved by presetting a critical level with an APP software. Such a combination of highly sensitive and selective detection, real-time online operation, and simple readouts, along with the easy construction, makes the proposed microchip platforms ideal candidates for further investigations and applications. The strategy of the ultrasensitive microchip integrated with smart microgel presented here circumvents the difficulties in simultaneously reducing the detection limit and improving the easy-to-operate property of detection techniques for trace analytes. It can be used to construct versatile new detection platforms for real-time detection of various kinds of trace analyte signals just by incorporating other stimuli-responsive microgels (6, 24–28), which might be a fertile area of research. Due to the excellent ultrasensitive, fast, and easy-to-operate properties, the detection platforms equipped with microchips incorporating smart microgel will provide ever-better performances in myriad applications including environmental protection, disease diagnosis and epidemic prevention, and may open up new areas of application for hydrogel-based detection techniques.

Materials and Methods

In Situ Synthesis of Cylinder-Shaped Microgel Within Glass Capillary.

The uniform cylinder-shaped poly(N-isopropylacrylamide-co-benzo-18-crown-6-acrylamide) [P(NIPAM-co-B18C6Am)] microgel is in situ synthesized within a glass capillary microchannel by developing an advanced rotation-based UV-irradiation method. For synthesis of the cylinder-shaped microgel, typically, 2 mL aqueous solution containing 0.3 mmol monomer benzo-18-crown-6-acrylamide (B18C6Am) (TCI) and 2.0 mmol monomer N-isopropylacrylamide (NIPAM) (TCI), 0.04 mmol cross-linker N,N′-methylene-bis-acrylamide (Chengdu Kelong Chemicals) and 0.037 mmol photoinitiator 2,2′-azobis(2-amidi-nopropane dihydrochloride) (TCI) is injected into a glass capillary, with inner diameter of 250 μm and outer diameter of 960 μm. The mole ratio of crown ether to NIPAM is set at 3:20 in our work, because the as-prepared microgel shows good response performance with the mole ratio of crown ether to NIPAM at 3:20, and further increase of the mole ratio shows little effect in improving the response performance (19). The monomer-solution-loaded capillary, with one end fixed on a rotating motor, is inserted into two stainless steel tubes with inner diameter of 1,000 μm. The stainless steel tubes are placed on a hot and cold stage (mk1000, Instec) to keep the synthesis temperature at 0 °C. The exposed part of capillary between the two steel tubes is covered by a patterned mask, with a transparent rectangular area (size: 130 μm × 1 cm) for UV irradiation. Then, the monomer-solution-loaded capillary is rotated at a speed of 60 rpm and irradiated with UV light (λ = 365 nm) for 120 s to in situ synthesize a uniform cylinder-shaped microgel inside the capillary (Movie S1). Compared with the traditional one-direction UV-irradiation method that usually leads to nonuniform cylinder-shaped microgel (SI Appendix, Fig. S1 A and B), such a rotation-based UV-irradiation method ensures 360° uniform UV irradiation for fabricating microgel with uniform cylinder shape (SI Appendix, Fig. S1 C and D).

Construction of Pb²⁺ Detection Platform.

After the UV-initiated synthesis of the microgel inside capillary, the microgel-incorporated capillary is used for assembly of the Pb²⁺ detection microfluidic chip (SI Appendix, Fig. S2). First, the microgel-incorporated capillary is fixed on a glass slide by epoxy resin, and clamped by two fixtures for fixation (SI Appendix, Fig. S2 A and B). Then, another capillary, with inner diameter of 170 μm and outer diameter of 960 μm, is placed into the interstice between the two fixtures and fixed on the glass slide for coaxial alignment with the first capillary (SI Appendix, Fig. S2C). After that, a cylinder bar of stainless steel with diameter of 165 μm is coaxially inserted into the capillaries to support the microgel, followed with fixation of the cylinder bar on the glass slide and seal of the second capillary by epoxy resin, and finally removal of the fixtures (SI Appendix, Fig. S2 D and E). The coaxiality of the cylinder bar, glass capillary, and cylinder-shaped microgel is important for the microgel fixation, because uncoaxial alignment of the microgel and cylinder bar leads to the lean of the microgel under flow impact. After the assembly, the unpolymerized solution in the capillary is removed, and thus the microgel-based Pb²⁺ sensor is obtained. Next, two needles are, respectively, connected to the two ends of the microgel-incorporated capillary, and sealed with epoxy resin as the inlet and outlet for fabricating the microfluidic detection chip (SI Appendix, Fig. S3A). Finally, the outlet of the detection chip is connected to a flowmeter of microfluidic control system (FLU_L, Fluigent) by polyethylene pipes for constructing the Pb²⁺ detection platform (SI Appendix, Fig. S3B). The dynamic swelling behaviors of the cylinder-shaped microgels with different sizes are studied by transferring the microgel from pure water to 10⁻⁵ M Pb²⁺ solution and measuring its dynamic size change using optical microscope (BX61, Olympus). The calculated values of τ_v for the microgels with different sizes are obtained from equation τ_v = r²/(π²×D) (23), based on D derived from the dynamic swelling behaviors.

Determination of the Optimal Operation Temperature for the Detection Platform.

To determine the optimal operation temperature for the Pb²⁺ detection, effects of temperature and Pb²⁺ concentration ([Pb²⁺]) on the flow rates are studied. First, pure water is supplied into the detection platform at a certain temperature. Then, Pb²⁺ solution with a certain concentration is supplied into the detection platform at the same temperature as that of pure water. Both pure water and Pb²⁺ solution are supplied under a constant pressure of 30 kPa by the microfluidic control system. The equilibrated flow rates of pure water and Pb²⁺ solution after being supplied for 15 min are measured by the flowmeter for evaluating the equilibrated flow rate changes (ΔJ). During the experiments, the temperature of pure water and Pb²⁺ solution is varied from 30 °C to 40 °C, and [Pb²⁺] is varied from 10⁻⁹ M to 10⁻⁴ M. The temperature at which the detection platform shows the most significant ΔJ value is defined as the optimal operation temperature.

Test of the Response Time and Sensitivity of the Detection Platform.

The response time and sensitivity of the detection platform are investigated by monitoring the dynamic change of flow rates after switching pure water to Pb²⁺ solutions with different concentrations from 10⁻¹⁰ M to 10⁻² M.

Test of the Selectivity of the Detection Platform.

The selectivity of the detection platform is first investigated by monitoring the dynamic change of flow rates after switching pure water to Pb²⁺ solution as well as solutions containing other interfering ions with different concentrations from 10⁻¹⁰ M to 10⁻³ M. Then, the equilibrated flow rate changes (ΔJ) after switching pure water to solution that concurrently contains Pb²⁺ and interfering ions for 15 min are studied. The interfering ions include Ba²⁺, Sr²⁺, K⁺, and Na⁺, each with concentration 1∼1,000× as large as the [Pb²⁺]. All of the solutions are flowed at 34 °C under a constant pressure of 30 kPa.

Test of the Repeatability of Detection Platform.

The repeatability of detection platform for Pb²⁺ detection is investigated after alternatively and repeatedly injecting pure water at 55 °C and 25 °C into the detection platform, each for 3 min, for Pb²⁺ removal. The Pb²⁺ removal performance after different wash cycles is estimated by measuring the flux recovery ratio (R_F). After complete Pb²⁺ removal (R_F = 100%), the detection platform is used for repeated Pb²⁺ detection (SI Appendix, Fig. S7).