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Synthesis of Monodisperse Plasmonic Magnetic Microbeads and Their Application in Ultrasensitive Detection of Biomolecules Chao Yuan, Yunte Deng, Xuemeng Li, Chengfei Li, Zhidong Xiao, and Zhuang Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01510 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018
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Analytical Chemistry
Synthesis of Monodisperse Plasmonic Magnetic Microbeads and Their Application in Ultrasensitive Detection of Biomolecules Chao Yuan,†,ǁ Yunte Deng,‡,ǁ Xuemeng Li,† Chengfei Li,† Zhidong Xiao,*,† Zhuang Liu*,§ †
College of Science, Huazhong Agricultural University, Wuhan 430070, China Department of Pathology, Hubei Cancer Hospital, Wuhan, 430079, China § Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, China ‡
ABSTRACT: Plasmon-enhanced fluorescence (PEF) based analytical technology has recently demonstrated its ability in detecting biomarkers with ultrahigh sensitivity. However, the scope of the PEF-based technology has been hindered by its reliance on flat substrates with relatively low binding kinetics and the limited multiplex detection ability. Herein, we reported a simple yet robust method for the fabrication of plasmonic magnetic microbeads (PMMBs)-based suspension array technology (SAT) with fluorescence enhancement of about 60-fold, improving detection limit of biomarkers by 2-order of magnitude towards 100 fM. We also demonstrated the performance of this method for the detection of anti-acidic ribosomal phosphoprotein 0 (anti-P0) autoantibody in sera from systemic lupus erythematosus (SLE) patients. Owing to the high sensitivity and efficient magnet-based sample collection, our method can be employed for detection of ultra-small volume of samples (e.g. 2 µL), promising for point-of-care detection. Furthermore, a size-encoded PMMBs-based multiplexed suspension array for simultaneous detection of multiple biomarkers is realized, illustrating the great potential of this technology in high-throughput disease diagnosis applications.
giving a great push to the development of PEF-based technology towards clinic research.17,18 Suspension array technology (SAT), a platform constructed by plugging microbeads into flow cytometry system, offers a new approach for large-scale screening applications. Suspension array is widely considered as the evolution of planar microarray due to its advantage in faster binding kinetics, lower sample volume needed, and multiplexing detection ability by optically encoding microbeads with combinations of fluorescent materials and decoding with flow cytometry.26-29 However, one important limitation for SAT is its relatively low detection sensitivity due to the limited number of fluorescence dyes bound to one bead. Thus, improving the detection sensitivity is always of great importance for further improvement of the SAT. Despite the successful integration of PEF mechanism into planar microarrays for bio-sensing applications, PEFbased SAT bioassay platform, to the best of our knowledge, has been relatively less explored thus far until a few recent reports, in which gold-coated glass beads were developed for SAT-based bio-detection.19,30,31 However, the practical applications of the reported PEF-SAT would be hindered by complicated sample preparation procedures and tedious centrifugation steps for collection of beads. During this process, the inefficient recovery of microbeads would become a problem especially for detection of very small amounts of samples. In this study, we develop a robust method for the fabrication of highly uniform plasmonic magnetic microbeads (PMMBs) featured with metal nano-island coverage for the
INTRODUCTION Plasmon-enhanced fluorescence (PEF), a physical phenomenon firstly reported for organic fluorophores located at nanometric distances from silver nanostructure by Lakowicz,1 can dramatically increase the quantum yields and photostability of fluorescent molecules. Therefore, PEF has been widely recognized as an attractive bio-sensing technique with improved sensitivity and resolution in practical applications.2-14 To date, dominant PEF-based biosensors mainly focus on deposition of gold nano-island membrane on glass substrates due to their excellent signal uniformity over large areas, easy fabrication route and stability over time. Such methods have demonstrated their potential in the analytical chemistry community with the possibility of detecting biomolecules with ultrahigh sensitivity.15-25 The physical principles behind this PEF-based solid microarray assays involve an enhancement in excitation field strength, reduction in excited state lifetime and overall apparent increase in fluorescence quantum yields of the used fluorescent dyes, leading to sharp increase of the signal-to-noise ratios over standard protein microarrays.15 For example, plasmonic gold film based microarray with near-infrared fluorescence enhancement of up to 100-fold has been reported for the cancer biomarker detection with fM sensitivity.20 Strikingly, PEF-based planar chip recently has displayed its superiority in diagnosis of diabetes and Zika virus with satisfactory results. More importantly, unknown biomarkers discovery can be realized by this method due to its high sensitivity and specificity,
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optimal plasmon-enhanced fluorescence, strong magnetism that allows efficient collection of beads, as well as varied sizes for multiplex detection of multiple targets simultaneously (Scheme 1). The synthetic route for PMMBs involves four steps, including surface modification of polystyrene (PS) microbeads, electrostatic adsorption of magnetic Fe3O4 nanoparticles (NPs), polydopamine (pDA) shell coating, and gold nanoparticles-induced silver nano-island membrane coating. It is worth mentioning that the multiple steps for PS microbeads coating are performed simply by solution reactions at room temperature, and could be easily scaled up. Owing to the highly uniform coverage of silver nano-island membrane on those magnetic microbeads, a high fluorescence enhancement factor of about 60-fold for fluorescein CF647 could be achieved. Compared with commercially available PS microbeads, our prepared PMMBs-based suspension array affords a broad dynamic range and improved detection sensitivity by approximately two orders of magnitude towards 100 fM for biomarker carcinoembryonic antigen (CEA), only one order of magnitude lower than the sensitivity of substrate-based PEF method reported.20 Importantly, the detection results obtained by our platform show excellent agreement with the clinic data for the detection of anti-acidic ribosomal phosphoprotein 0 (anti-P0) autoantibody in sera from systemic lupus erythematosus (SLE) patients. Finally, this PMMBs platform is expanded to multiplexed detection by using size-encode PMMBs for three biomarkers prostate-specific antigen (PSA), carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) with similar sensitivities. Our results collectively demonstrate the great promises of the magnetic PEF-based suspension array platform for bioassay with improved sensitivity in practical applications.
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and could be resuspended well in solution without aggregation simply by pipette mixing after magnetic separation. Next, the prepared magnetic microbeads were coated with a thin pDA shell by incubating them with dopamine in tris buffer (pH=8.5) to immobilize the attached magnetic Fe3O4 NPs. The obtained black magnetic microbeads showed a dark gray color, indicating the dopamine polymerization on their surface (Figure 1d). Moreover, the TEM image of dopaminetreated magnetic microbeads revealed the existence of a pDA shell with a thickness of about 50 nm (upper left insert in Figure 1d). Most importantly, pDA shell-coated magnetic microbeads dramatically enhanced the solubility of bare Fe3O4 NPs-coated microbeads, and thus improved the magnetic separation efficiency and shortened the magnetic response time. As revealed by the upper right insert in Figure 1d, pDA-coated magnetic microbeads could be rapidly attracted to the inner wall of the glass bottle with almost 100% collection yield in less than 2 min, while a fraction of magnetic microbeads would float on the water in the case of pure Fe3O4 NPs-coated microbeads (upper right insert in Figure 1c). Another reason for pDA coating is that Au NPs could be readily adsorbed on the surface of those beads without the need of any additional treatment, due to the presence of abundant amine groups on the pDA shell.32 For the following noble metal nano-island membrane coating, 13 nm Au NPs were directly adsorbed onto the surface of pDA-coated magnetic microbeads, followed by the addition of silver-ammonia growth solution in the presence of glucose alkali solution as the reducing agent. In order to obtain silver nanoisland-based plasmonic microbeads with the best fluorescence enhancement performance, the size of silver NPs and the nanogap between adjacent NPs should be precisely controlled. Scanning electron microscopy (SEM) images (Figure 1e) were taken to observe the surface topography of PMMBs synthesized by adding different volumes of silver growth solutions. After silver coating, those microbeads were densely and uniformly coated with elongated tortuous silver nano-islands. As the increase of added silver growth solution volume, increased silver nano-island size and thickness, together with the reduction of inter-nanoisland gaps were observed. However, when the growth solution further increased, the silver nano-islands eventually coalesced into a continuous film, along with the formation of free silver NPs in the reaction solution (Figure S2 in Supporting Information). It is known that the silver nano-island size/shape and the inter-nano-island gap spacing would play a vital role in obtaining optimal fluorescence enhancement in bioassay. Next, we examined the fluorescence enhancement effect of the asprepared PMMBs with various surface silver nano-island membrane by incubating cyanine-based far-red fluorescent dye CF647-labelled avidin with PMMBs in PBS solution for 2 h in the dark prior to flow cytometry measurement. Carboxylcoated PS microbeads with the same diameter and bead number were used as the control. For the quantification of avidin absorbed on the surface of PMMBs and PS microbeads, the avidin-coated microbeads were treated by biotin-labelled HPR, followed by the addition of TMB substrate solution. The optical density of the solution could reflect the abundance of avidin absorbed on the surface of microbeads. The fluorescence enhancement factor (E) of PMMBs was defined by E =
RESULTS AND DISCUSSION Fabrication of PMMBs. In this work, uniform PS microbeads were chosen as the host material for the fabrication of PMMBs using a layer-by-layer method (Figure 1a). Initially, the synthesized PVP-coated PS microbeads were sulfonated by a mixture of concentrated sulfuric acid and acetic acid to render their surface negatively-charged. For the immobilization of negatively-charged citrated-capped Fe3O4 NPs on the surface of PS microbeads, the microbeads were firstly treated with polyelectrolyte PDAMAC to render their surface positivelycharged. As revealed by the zeta potential results, the surface charge of sulfonated PS microbeads changed from -48.5 to +15.2 mV after PDAMAC treatment, confirming the successful surface modification of the PS microbeads (Figure S1 in Supporting Information). After incubation with negativelycharged Fe3O4 NPs, the surface of PS microbeads were densely coated with magnetic NPs, as revealed by the scanning electron microscopy (SEM) images of those beads before and after Fe3O4 NPs immobilization (Figure 1b and c). In addition, the obtained microbeads changed from white to yellowish-brown color, and could be separated from the solution simply by an external magnet, confirming the successful immobilization of Fe3O4 NPs on their surface (upper right inserts in Figure 1b and c). Moreover, transmission electron microscopy (TEM) image of the magnetic-responsive microbeads further confirmed the existence of Fe3O4 NPs shell on the surface of microbeads (upper left insert in Figure 1c). It should be noted that the obtained magnetic microbeads are superparamagnetic
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Analytical Chemistry
(IPMM·APMM)/(IPS·APS), where IPMM and IPS are the fluorescence intensity of PMMBs and PS microbeads collected from flow cytometry, and APMM and APS represent the optical density of TBM substrate solution at the wavelength of 480 nm after treatment with PMMBs and PS microbeads. Figure 1f shows the flow cytometry data of PMMBs synthesized from 35 µL of silver-ammonia solution and PS microbeads with the same beads number (4*107). The fluorescence intensity of PMMBs was about 20 times higher than that of PS microbeads. Given the number of avidin on the surface of PS microbeads was about 3 times as much as that on PMMBs (Table S1 in Supporting Information), it is concluded that PMMBs prepared under this condition could afford as much as 62-fold enhancement for dye CF647, which is the maximum of enhancement factor over other PMMBs synthesized by adding different amounts of silver growth solution (Figure 1g). This huge enhancement factor is attributed to the combination of strong electromagnetic field enhancement from the nano-gaps between silver nano-islands and resonance coupling of CF647 fluorescence emission with the surface plasmon in the underlying silver nano-island membrane.20
microbeads method for the quantification of CEA. In addition, by taking advantage of the magnetism of our PMMBs, the time for each cycle of PMMBs separation could be dramatically shortened to less than 2 min with the help of magnet. More importantly, the decrease of biomolecules activity and the risk of losing beads caused by repeated centrifugation steps could be dramatically eliminated by magnetic separation, thus improving the reproducibility and reliability of the detection results. The confocal fluorescence microscope images vividly illustrated the strong and uniformly distributed fluorescence signals on PMMBs after being incubated with CEA and recognized by CF647-labelled CEA detection antibody (Figure 2e). Moreover, it should be noted that the estimated number of capture antibody conjugated on each PMMBs and PS microbeads was almost the same using BCA method, further confirming the plasmon-induced fluorescence enhancement effect on the surface of our PMMBs. To evaluate the performance of PMMBs method for practical use to detect clinical samples, 10 clinical human serum samples from Hubei Cancer Hospital of China were collected and used for a double-blind experiment. The concentrations of CEA were pre-determined by the standard ELISA method in those samples but kept unknown until their values were also determined by our PMMBs-based bioassay method. In our work, we firstly investigated whether higher CEA concentrations in undiluted sera would correlate well with higher fluorescence signal intensities of PMMBs, a key factor determining the interference of other existing biomolecules within those samples to our method. As shown in Table 1, CEA concentrations in sera and fluorescence intensities of PMMBs exhibited positive correlation. To further test the detection sensitivity of the PMMBs method to lower CEA concentrations, we used 50-fold FBS-diluted serum samples to repeat the experiment, and the results showed that the fluorescence signals of PMMBs could still be clearly distinguished from that of noise. Importantly, the concentrations of CEA obtained from PMMBs-method showed great consistence with those obtained by the ELISA method, indicating the ability of our PMMBs platform for CEA detection in clinical samples with great sensitivity and accuracy. Moreover, the CEA detection using our PMMBs-based suspension array method requires much less sample (2 µL) than the classic ELISA method (mL level).
Carcinoembryonic antigen (CEA) assay on PMMBs. To demonstrate the performance of PMMBs in bioassay, carcinoembryonic antigen (CEA), an important cancer biomarker, was used as the target protein (Figure 2a). PMMBs were first coated with 3-mercaptopropionic acid and then activated by 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS) for the conjugation of NH2-PEGCOOH molecules to increase the hydrophilicity of PMMBs in phosphate buffered saline (PBS) and to decrease the possibility of the direct contact of CF647 on the surface of silver NPs, which could result in the fluorescence quenching of the dye. After blocking with glycine, the PEG-COOH-capped PMMBs was activated again by EDC/NHS, followed by CEA capture antibody conjugation and glycine blocking to obtain CEAspecific PMMBs probe. Commercially available 4-µm carboxyl-coated PS microbeads were also conjugated with anti-CEA antibody by the same EDC/NHS method and used as the control. After incubating anti-CEA conjugated PMMBs or PS microbeads with different concentrations of CEA (in the range of 1 nM to 100 fM), CF647-labelled CEA detection antibody was used as fluorescent reporter to recognize CEA captured on the surface of those microbeads. Magnetic separation was carried out to collect PMMBs while centrifugation was used to collect PS microbeads after each step of incubation. Flow cytometry was then used to determine the fluorescence intensities of those microbeads (Figure 2b-d). As shown in the flow cytometry histograms (Figure 2c&d), with the increase of CEA concentration from 100 fM to 1 nM, the fluorescence intensity of PMMBs gradually increased and finally reached to about 560-fold stronger than that of the blank beads. Even at the concentration of 100 fM, about 4-fold enhanced fluorescence was still observed. In contrast, when the conventional PS microbeads were used for CEA capture and detection, there was no obvious CEA signals at or below the CEA concentration of 1 pM (Figure 2b&c). Therefore, our PMMBs demonstrated significantly improved detection sensitivity by approximately two orders of magnitude and a broader dynamic range than non-plasmonic PS
Autoantibody detection for systemic lupus erythematosus (SLE) diagnosis. SLE, an autoimmune disease of unknown etiology, can affect multiple organ systems with variable severity.33-35 Among the various autoantibodies found in SLE, anti-acidic ribosomal phosphoprotein 0 (anti-P0) antibody has shown promise to predict disease activity.36,37 Next, we used our PMMBs-based method to detect anti-P0 in clinical serum samples. Ten systemic lupus erythematosus (SLE) serum samples (2 anti-P0 autoantibody positive and 8 negative) were used to test the performance of our method also in a doubleblind experiment. Unlike the surface functionalization of PMMBs used in CEA detection, protein antigen P0 was immobilized onto the surface of PMMBs by nonspecific adsorption, followed by BSA blocking. CF647-labelled goat-anti human IgG was used as fluorescent reporter to evaluate the sensitivity of P0-coated PMMBs to anti-P0 autoantibody in
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serum (Figure 3a). As shown in Figure 3b, according to the clinical data, if a patient’s serum contained anti-P0 antibody (sample #4 and #5), a strong fluorescence signal on PMMBs was detected from 1:50 FBS-diluted serum with total volume of 100 µL. In contrast, the fluorescence signals were very weak in all of the anti-P0 antibody negative sera with the cutoff value of 200, demonstrating the accuracy of our method for anti-P0 auto-antibody detection in SLE clinical serum samples.
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detection of our PEF-based suspension microarray could dramatically expanded without the compromise of sensitivity by coupling PEF technology with dye-encoded microbeads system. CONCLUSIONS In summary, we have introduced PMMBs with highly-uniform and tunable sizes as a unique SAT technology platform. The PMMBs platform affords a fluorescence enhancement of ~60fold for red fluorescent dye CF647 and as low as 100 fM sensitivity for CEA detection, an almost 2-orders of magnitude improvement compared with that of conventional PS microbeads. Moreover, the applications of PMMBs-based suspension array for the detection of cancer biomarker CEA and anti-P0 autoantibody in sera from patients have been successfully demonstrated by double-blind experiments, showing excellent reliability as compared with clinical data. By employing PMMBs with three different sizes, multiplexing detection of three types of biomarkers (CEA, PSA and AFP) can be realized in serum samples with high accuracy. Our bottom up synthesis procedure of PMMBs-based suspension array is scalable, simple and suitable for automation. Compared to conventional microbeads-based SAT technology relying on centrifugation for beads collection, our PMMBs could be easily separated by a magnet within 2 min with nearly 100% recovery yield. Importantly, our method only requires 2 µL sample of serum, demonstrating the possibility of point-ofcare disease diagnosis. The multiplexing ability of our PMMBs-based platform would be of particular attractive for high-throughput detection of multiple targets within complex samples. We thus believe this PEF-based SAT based on PMMBs could be employed as the next generation of suspension array in biomarker detection and disease diagnosis with improved sensitivity in a multiplexed manner.
Multiplexing detection of biomarkers by size-encoded PMMBs. The sizes of PMMBs could be determined by the diameters of initial PS microbeads. Therefore, our method could be expanded to the synthesis of PMMBs with controllable sizes. To demonstrate the multiplexing capabilities of PMMBs-based suspension array, PMMBs with three different diameters (3 µm, 4 µm and 6 µm) were used for the simultaneous detection of three kinds of biomarkers (CEA, PSA and AFP). PMMBs with diameter of 3 µm and 6 µm were synthesized using the same method for 4 µm PMMBs, and their SEM images were shown in Supporting Information Figure S3. Commercially available Carboxyl-coated PS microbeads (3 µm and 6 µm) were used as the controls to evaluate the fluorescence enhancement effect of the prepared 3 µm and 6 µm PMMBs, respectively. The enhancement factors of ~60-fold for both two types of PMMBs compared to their respective PS beads could also be obtained by controlling the added silver growth solution (Supporting Information Figure S4). Notably, the three types of PMMBs with different sizes could be clearly distinguished by the flow cytometry due to the direct correlation between the bead size and front scattering, thus enabling the quantification of fluorescence signals for different sizes of beads without obvious cross-talk (Figure 4a). For the multiplexing detection, 3 µm and 6 µm PMMBs were functionalized with PEG-COOH and then conjugated with PSA and AFP capture antibody, respectively, using the same method for CEA capture antibody-conjugated 4 µm PMMBs. Similarly, the sensitivities for the separated detection of PSA or AFP biomarkers could also be improved by two orders of magnitude as compared with the bare PS microbeads (Figure 4b&c). Next, the multiplexing detection was carried out after incubating the mixture of three types of PMMBs (4*105 beads for each size) with fetal bovine serum (FBS) spiked with three types of biomarkers (CEA, PSA and AFP) at different concentrations. Those beads were then washed with PBS containing BSA (0.5%) and then incubated three types of CF647-labelled detection antibodies. Flow cytometry was then employed to measure fluorescence intensities for three types of beads, which were separated based on their front scattering signals that correlated to the bead sizes. As shown in Figure 4d, the concentrations of those three biomarkers could be accurately detected with recoveries ranging from 88% to 107%, demonstrating the potential of our PMMBs-based method for the multiplexing detection of biomarkers in serum samples with high sensitivity and accuracy. In addition, as the commercialized suspension microarray technology owned by Luminex Corporation has already shown its unprecedented encoding capacity (up to 100 bead sets with unique spectral addresses) by internal incorporating PS microbeads with two spectrally distinct fluorescent dyes, the capacity of multiplex
EXPERIMENTAL SECTION Chemicals and materials. Styrene, poly(vinypyrrolidone) (PVP, Mw: 55000), poly(diallydimethyl) ammonium chloride (PDAMAC, MW: 150000), 2, 2’-azobis-(2methylbutyronitrile) (AMBN), glucose, avidin, Carboxylpoly(ethylene glycol)-amine (COOH-PEG-NH2, 20KD), CF647-NHS ester, biotin-NHS ester, tris (2-carboxyethyl) phosphine hydrochloride (TCEP), Tris-HCl, dopamine hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), fetal bovine serum (FBS), BCA Assay Kit, PSA and rabbit polyclonal anti-PSA antibody were purchased from Sigma Aldrich. COOH-coated polystyrene (PS) microbeads (3 µm, 4 µm and 6 µm, CV
