Author's Accepted Manuscript
3D graphene nano-grid as a homogeneous protein distributor for Ultrasensitive biosensors Zhenyu Chu, Lei Shi, Wanqin Jin
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S0956-5663(14)00338-8 http://dx.doi.org/10.1016/j.bios.2014.05.006 BIOS6767
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Biosensors and Bioelectronics
Received date: 10 March 2014 Revised date: 28 April 2014 Accepted date: 2 May 2014 Cite this article as: Zhenyu Chu, Lei Shi, Wanqin Jin, 3D graphene nano-grid as a homogeneous protein distributor for Ultrasensitive biosensors, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2014.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
3D graphene nano-grid as a homogeneous protein distributor for ultrasensitive biosensors Zhenyu Chu, Lei Shi and Wanqin Jin* State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical engineering, Nanjing Tech University, 5 Xinmofan Road, Nanjing 210009, P. R. China. Corresponding Author * E-mail: [email protected]. Fax: +86 25-8317-2292; Tel: +86 25-8317-2266. Abstract: In order to realize the protein uniform immobilization, a 3D nano-gird architecture of thiol grafted graphene film was fabricated to serve as a novel linker between protein and substrate. Relied on the online monitor by QCM, graphene deposition process can be exactly controlled to construct the perfect and continuous cavities with the consistent size of 500 nm. The synergetic characterization of FESEM and Nano-indentation characterizations have revealed the strong stability of grid structure to provide a firm foundation for further protein adsorption. Instead of common partial aggregation behavior, proteins can be spontaneously distributed into cavities by the interaction from thiol group. According to the verifications of various proteins, the efficiency of this distributor will not be constricted by the category and amount of protein, which exhibit its versatility of homogeneous distribution. Glucose and lactate oxidase loaded graphene distributors were directly served as biosensors to verify the superiority of distribution. Their sensitivities can be remarkably improved three times since the adoption of this nano-grid structured graphene distributor.
Keywords: Graphene film; In-situ synthesis; 3D nano-grid; Various proteins immobilization; Ultrasensitive biosensors 1. INTRODUCTION Generally, protein immobilization is an essential stage referred to the bio-materials fabrication (Chen et al, 2001), fermentation process design (Doran et al, 1986) and biosensor development (Mirsky et al, 1997). The immobilization status of protein, such as morphology, distribution and accumulation style, will synergistically affect the performance of bio-reaction process (Hudson et al, 2008; Onoda et al, 2012). Normally, the combination between protein and support which produces a weak interaction on interface is depended on a cross-linking reagent to produce a bridge function (Zhang et al, 2007; Niemeyer et al, 2001; Delamarche et al, 1997). It is traditionally consisted by the high viscous polymer to serve as a “glue” for protein adhesion. Hence, the control of protein distribution is difficult due to the rarely uniform coverage of cross-linker on substrate surface. In order to realize the regular distribution, only relying on the uncontrollable “glue” is useless. Therefore, a novel linker which is required to be of excellent biocompatibility, thermal stability and mass transfer ability should be introduced to build a strong chemical interaction of interface between protein and substrate. Besides, with the purpose of regular protein distribution, the structure of this linking layer should also possess a uniform microstructure to induce the protein settling down. Graphene has aroused the numerous attentions of various scientific fields because of its abundant unique properties. Its high surface area, rapid conductive rate and good environment stability are most encouraged to serve as superior advantages in bio-material fabrication (Patil et al, 2009; Song et al, 2010; Dong et al, 2011). However, because of the sole sheet shape, graphene morphology of formed film most keeps the layered accumulation. More structures of graphene films are difficult to be created. According to recent literatures, three dimensional (3D) graphene films which were
normally prepared by the reduction of graphene oxide have been confirmed to own higher performance due to the larger contact area and easy device construction (Hu et al, 2013; Dong et al, 2012). But the cavity size and location of these structures are still random formed which cannot provide a uniform and proper space for protein loading. The uniform distribution of protein can effectively avoid the partial aggregation behavior which is benefited to obviously increase the contact area and reduce the mass transfer resistance. Therefore if a homogeneous distribution to protein is expected to realize, the key is realization of a highly uniform structure of graphene for the protein immobilization.
Figure 1. The synthesis process scheme of graphene nano-grid based distributor.
In this work, thiol grafted graphene was adopted as a linking substance to produce a bridge function between protein and substrate. Relied on an online monitor strategy, the graphene layer was accurately shaped to a 3D nano-grid structure with a numerous 500 nm cavities by nanolithography approaches. Various proteins, glucose oxidase, lactate oxidase and bovine serum albumin, were respectively tested to verify the versatility of this protein uniform distributor. Based on the chemical interactions of thiol group belonged to graphene, proteins can be not only stably adhered on metal surface, but also uniformly and regularly distributed into the cavities of graphene grid. Moreover, glucose and lactate oxidase loaded protein distributors have be directly served as highly sensitive biosensors to exhibit the advance features of homogeneous protein distributors.
2. EXPERIMETAL 2.1 Reagents and apparatus Thiol graphene (4.0 wt% hydroxyl ratio) was synthesized by Jcnano technology Co., Polystyrene latex microspheres (PS, 500 nm, 2.5 wt% in water) were purchased from Alfa-Aesar
company.
K4[Fe(CN)6]·3H2O
(Sigma-Aldrich),
FeCl3·6H2O
(Sigma-Aldrich), (3-glycidyloxypropyl) trimethoxysilane (Sigma-Aldrich), sodium n-dodecyl sulfate (Alfa-Aesar), sodium L-lactate (Alfa-Aesar), glucose (Sinopharm Chemical Reagent Co., Ltd, China) and 30 wt% H2O2 (Sinopharm Chemical Reagent Co., Ltd, China) were of analytical grade purity and used without further purification. 4.5 mg glucose oxidase (GOD, EC 1.1.3.4, 168800 units per g, from Aspergillus niger, Sigma-Aldrich) and Bovine Serum Albumin (BSA, Sigma-Aldrich) were respectively dispersed in 1500 μL of 0.05 phosphate buffer solution with 0.1 M KCl (PBS, pH=6.5) and was sufficient for preparation of three electrodes. 2.5 mg lactate oxidase (LOD, EC 1.13.12.4, 20 units per mg, from Pediococcus sp., Sigma-Aldrich) was dispersed in 500 μL PBS and separated into 25 separate vials (2 units per 20 μL). All solutions were prepared with deionized water. Electrochemical characterizations were realized by an electrochemical workstation (CHI 660C, Shanghai Chenhua, China). All cyclic voltammetry (CV) experiments were operated in PBS at 25℃. A Pt wire and Ag/AgCl were used as the counter and reference electrodes, respectively. The scan rate was 50 mV s-1. Electrochemical Impedance Spectroscopy (EIS) characterization was carried out at the -0.05 V potential. The measurement of the voltage amplitude of 5 mV from the frequency range 0.05 Hz to 105 Hz was made in the buffer solution. Online monitoring of graphene coverage was fulfilled by quartz crystal microbalance (QCM200, Stanford Research Systems, Inc, USA). All field emission scanning electron microscopy images (FESEM) were taken by Hitachi-4800. The mico-structure of graphene was detected by high-resolution transmission electron microscopy (HRTEM) (JEM-2010 UHR, Japan). Prior to the
HRTEM measurements, the graphene film was scraped from the electrode surface, ultrasonically dispersed in ethanol. Nano-indentation characterization was operated on NanoTest Vantage (Micro Materials, UK). 2.2 Pretreatment of the Au disk In order to satisfy the clean and plane demands to substrate, Au disk (2 mm diameter) was first polished as a mirror by metallographical sand paper. Then the disk was dipped into a Piranha solution (7:3 mixture of H2SO4 and H2O2, v/v) for 30 min and rinsed with water. Finally, it was washed in ultrasonic environment for 30 min for ready usage. 2.3 Preparation of graphene nano-grid Since finishing above pretreatment step, one drop of PS beads was firstly added on Au disk which is enough to cover whole metal surface. The disk was held stationary for 1 min to enable sufficient combination of PS and Au. Secondly, the substrate was slowly immersed into deionized water, and then one or two droplets of 2% Sodium n-dodecyl sulfate were dropped into this water to change the surface tension which is aimed to disperse PS to form monolayer. Subsequently, the prepared monolayer of PS modified electrode was dried at 80 ˚C for 90 min in order to fix the beads to the surface. 5 mg thiol graphene powder was added into 20 ml ethanol to form graphene deposition source. In order to smash the graphene sheet to smaller size, this suspension liquid was then dispersed and vibrated by a probe-type sonicator (KBS-650, Kunshan Shumei, China) with a 20 KHZ of ultrasonic frequency for 30 min. Subsequently, the prepared monolayer PS/Au disk was vertically suspended to immerse the graphene source. During the deposition process, the liquid temperature was kept to 60 ˚C with stir in order to continuously condense the graphene source. After above step, disk was washed to clean the surface, and then moved to toluene for 30 min. Finally, the disk was dried in oven at 60 ˚C for 30 min.
2.3 Immobilization of proteins GOD, LOD and BSA were respectively applied as samples of protein. Graphene/Au disk was immersed into the divided enzyme solution for 1 hour. Then the disk was washed clean and ready for usage. 3. RESULTS AND DISCUSSION 3.1 Online control of graphene nano-grid formation
Figure 2. (a)The frequency and mass change of quartz crystal in graphene deposition process by QCM monitor; (b) to (d) is the structure evolution of graphene film surface respectively prepared under 200 s, 400 s and 600 s. (e) is the image under low magnification rate of (d); (g) to (h) are the HRTEM images of half-filled PS balls.
In order to build a 3D structure in graphene fabrication, as shown in Fig. 1, three steps are required to exactly fulfil. First step of template layout must ensure the regular and tight distribution of each PS nano-sphere. Subsequent process, graphene adsorption, is a key to the success of morphology construction. Amount of bonded graphene on Au surface will directly affect the integrity of final formed grid architecture. In this case, the accurate judgment of time node during graphene deposition is crucial. Hence, QCM technique was applied as an online strategy for the monitor of graphene adsorption
evolution. The whole graphene deposition process was detected by QCM equipment. The data of graphene amount shift on Au surface was online recorded. As shown in Fig. 2(a), total deposition process has happened two obvious adsorption stages: time periods before and after 500 s. Neglection of beginning instable process before ca. 100 s, the amount of graphene occurred a slow climb which indicated the initial combination of graphene on Au surface by strong Au-SH bond. It should be noticed that the space created by the neighbouring spheres is so narrow that only tiny graphene sheet can pass to arrive on metal surface for initial adsorption. Then, at the time of 500 s, a sudden elevation occurred to illustrate a rapid adsorption behaviour of graphene. This is due to the concentrating deposition method we adopted. Within 60 ˚C ethanol environment, graphene will continuously undergo a concentration to accelerate the “wall” building to surround the spheres on already formed foundation. Subsequently, the deposition again reached a stable stage which belongs to a steady aggregation between graphene sheets. Further deposition will cause the over-thickness of graphene film to cover whole template surface (time of 800 s, S1) which affects the grid formation. Accordingly, the whole preparation of graphene around template can be considered termination beyond ca. 600 s. For directly confirming the morphology evolution, the template removed graphene disks prepared by different deposition time were characterized by FESEM. The deposition periods of 200, 400, 600 s were selected as the typical samples. Exhibited in Fig. 2 (b), initially, the first arrival graphene sheets were crossed through the space between each PS sphere to combine with Au support. This amount of graphene is low to only surround the very bottom of PS, hence, since the removal of PS, just leaving a thin film with round traces. With prolonging the adsorption time, the graphene “wall” was bricked by graphene accumulation. According to the side view image in Fig. S2, it shows that graphene has covered the bottom region and partial gaps between PS balls. However, it is obvious that there are still some deficiencies to reveal in Fig. 2(c). Since
reaching a half-filled stage, we finally obtained an integrated and smooth grid of single layer structure with regular and uniform 3D nano-cavities of ca. 500 nm (Fig. 2(d)). Moreover, instead of just small region, a very large area of support can be covered with perfect nano-grid graphene film in Fig. 2(e). With the purpose to investigate the growth mechanism of grid wall, the graphene accumulation behavior was magnified under a HRTEM observation. The film of graphene covered PS spheres was peeled from Au surface, and dispersed in ethanol solution for TEM characterization. Based on the results in Fig. 2(f) to (h), it can be illustrated that multiple layers of films were covered on the sphere surface. Each layer may be not the same length, but the total length which relied on the stack is still long and continuous without fault. Further focusing on each layer, the thickness is ca. 3.4 Å which matches the standard graphene parameter (Ohta et al, 2006). Above evidences can indicate that, after the initial base film formation, the construction of grid wall structure is built by the encompassment of graphene sheets on the surfaces of PS beads. Then, the PS template was removed to leave the cavities and connected graphene walls to exhibit grid morphology. CV characterization measurement was applied to investigate the catalysis and electron transfer abilities of graphene nano-grid (Fig. S3). Bare Au disk, PS template covered Au (PS/Au), graphene modified PS/Au and graphene nano-grid modified Au (GE/Au) were respectively characterized by CV in the 0.05 M [Fe(CN)6]4-/[Fe(CN)6]3solution. With the calculation of each CV curves, graphene nano-grid is of the lowest resistance. Accordingly, the formation of nano-grid structure owns the best performance of reaction rate and resistance. This is attributed to the regular construction of film which can provide a uniform environment and channel to arouse the advantages of graphene in catalysis and electron transport. EIS technique was also applied to obtain the resistance data before and after the grid formation. According to the simulation of scanning data by equivalent circuit, the interface resistance between graphene and Au were calculated as 462.4 and 1291.2 Ω before and after PS template removal. This result
is consistent with above potential difference from CV characterization. It is mainly due to the existence of PS which owns low conductivity among graphene layers. Since the reaction beginning, the transfer channel between graphene and Au will be occupied by a layer of PS beads to slow down the amount of delivered electron which weaken the received current signal. 3.2 Protein distribution by graphene nano-grid
Figure 3 Nano-indentation test of prepared graphene film: (a) is the diagram of applied indenting force; (b) is the digital photo of indention surface; (c) is the FESEM image of indented region of film surface.
Before the immobilization of protein, the stability of graphene grid should be tested to provide a reliable environment for adsorption. A common trouble on graphene film is that it will often roll to change the morphology without a firm force to be adhered on support surface (Yu et al, 2007). This is a big challenge to maintain the stability of protein immobilization and application. Therefore, a nano-indentation measurement was adopted to investigate the interaction between Au substrate and graphene film. One prepared graphene grid film was scratched by an increasing force (Fig. 3(a)) from a diamond probe. As shown in Fig. 3(b), the trace of ravine was deeper and more obvious with the strength enhancement till the film had been thoroughly broken into two parts. We selected the tip scratch where began to happen the crack to characterize the micro-morphology of film. According to the surface image in Fig. 3(c), it can be found that, except the crack, the continuity and integrity of film had been maintained. Holes
also kept its original shape and size instead of deformation caused by the loss of constraint. Besides, the fracture surface did not roll up which illustrated that the elastic deformation was conquered by the substrate binding force of Au and –SH group. Above evidences can demonstrate that the graphene film can preserve its perfect grid and hole structure to resist the external force by the firm interaction between film and substrate. Subsequently, the proteins loading efficiency of graphene nano-grid was checked. In order to testify the versatile function in protein distributions, three different proteins: GOD, LOD and BSA were served as samples. GOD and LOD both belong to the oxidase category, but loading amounts are very different in real application. Different with oxidase, BSA is one of serum albumin. Therefore, the capacities of graphene grid on amount and category can be tested. In the whole immobilization process, proteins kept free adsorption without other external forces or cross-linkers. As our expectation, proteins will be captured into the cavities by the attachment function of thiol groups from graphene wall. Hence, the bonding behaviors between enzyme and graphene are prerequisite. The characteristic group changes of graphene nano-grid which was loaded by GOD were characterized by FT-IR in Fig. S5. Two extra peaks generate in IR curve, one locates at 1543 cm-1 and another places at 1633 cm-1. They are the representative groups: amide I and amide II which is general existence in protein structure (Wu et al, 2010). In the IR result of only graphene nano-grid, at 804 cm-1, there is a weak peak from C-SH bending (Pham et al, 2013). But it has disappeared since GOD immobilization. This illustrates free C-SH group has participated in the reaction of protein loading. This combination is a typical reaction between protein and –SH grafted material which is depended on the redox production of –S-S– group from protein and –SH group modified substance (Wilkinson et al, 2004; Yiu et al, 2001; Ovsejevi et al, 2009). Hence, around the thiol graphene formed grid architecture, proteins are promising to be arranged along hemispherical shape.
Figure 4. FESEM images of GOD loaded (a) graphene nano-grid (b) graphene layered film. The bar of inset is 1μm. (c) and (d) are LOD and BSA loaded graphene nano-grid, respectively.
GOD is one of the most applied proteins in biological detection. In order to harvest a high performance, its distribution is normally required as uniform as possible. Here, as shown in Fig. 4(a), the whole GOD loaded film surface was extremely uniform. The profile of graphene grid was still clear to be distinguishable. According to the magnified image of inset, the space of original cavities was not only absolutely filled by GOD, but also formed continuously round bulges with 500 nm size. On the contrary, if grid structure was not adopted, protein would aggregate together without restriction, and this layer would be consisted by some separated, irregular and uneven GOD clusters with much larger size (Fig. 4(b)). Meanwhile, LOD which was only 1/15 amount of GOD in real usage was repeated to load on graphene surface. FESEM characterization indicated that except some unfilled cavities due to the fewer amounts, graphene grid can still distribute protein uniformly along its framework. Besides of enzyme, another category of protein, BSA, was also attempted. Under the same loading amount as GOD, the distribution behavior and final surface morphology were nearly consistent. Consequently, the fabricated graphene nano-grid can be of a generally homogeneous
distribution function to the protein immobilization. 3.3 Effects of protein homogeneous distributions
Figure 5. CV comparisons of graphene nano-grid based (a) glucose biosensor and (b) lactate biosensor before and after the respectively additions of 2 mM glucose and lactate
The superiority of graphene based enzyme biosensor is the direct electron channel between electrode and active center of enzyme (flavin adenine dinucletide, FAD) instead of the necessity of mediators in traditional fabrication (Shao et al, 2010). Therefore, the operation potential of graphene based biosensor is usually much lower. As shown in Fig. 5, since the addition of 2 mM target glucose into the detection system, an obvious signal increase happened at the potential of ca. -0.6 V. The same phenomenon was also occurred in the lactate detection. Above results illustrate that prepared graphene film owns a strong response to the oxidase reaction at a special potential, and the reacted electrons can fast transfer through graphene layer to electrode surface. Accordingly, in the following performance characterizations, the operation potentials were determined. The
effects
of
protein
homogeneous
distribution
were
studied
by
a
chronoamperometry measurement. Taking GOD and LOD as models, the bio-sensing performance of nano-grid structure was compared with layer structure (the film morphology
was
shown
in
Fig.
4(b)).
According
to
the
calculation
of
chronoamperometry data (Fig. 6(a)), for GOD loading, homogeneous distribution can reach a sensitivity of 35.06 μA·mM·cm-2, and random adsorption was only 5.12 μA·mM·cm-2. Similarly, LOD loaded grid architecture also exhibited a nearly four times exceedance than layered distribution (9.98 vs. 2.76 μA·mM·cm-2). The relative standard deviation (RSD) of prepared graphene nano-grid biosensor is 10.1%. The reusability of prepared graphene grid based electrode was also investigated. The same electrode which was used by chronoamperometry characterization was stored in fridge at 4 ℃, and then repeated to test after 10 days. The sensitivity can only decreased to 87% which illustrates the excellent reusability of grid film. This may be attributed to the strong interface interactions of enzyme/graphene and graphene/Au by –SH group. In order to realize
the
quantitative
evaluation
of
enzyme
immobilization
behavior,
a
Michaelis-Menten constant KM in enzyme kinetics was calculated. It can represent the substrate concentration at which the reaction rate is at half-maximum, and is an inverse measure of the substrate's affinity for the enzyme (Kamln et al, 1980). The calculated KM for graphene grid based glucose biosensor is 0.71 mM. Compared with other graphene based glucose biosensors in Tab. S1, the KM constant, sensitivity and operation potential are all superior to only graphene film, even better than other high catalytic material doped graphene films. This excellent performance is due to the uniform distribution of enzyme immobilization. It will avoid the instability of enzyme caused by random accumulation on partial sites, and also increase the contact area of enzyme for target to obviously enhance the response signal in detection. Moreover, due to the low application potential, as-prepared biosensor can own an outstanding anti-interference ability in the co-existence of ascorbic acid and uric acid (Fig. S7). Therefore, the homogeneous protein distribution brings a superior behavior of performance.
Figure 6. (a) The fitted diagram of GOD and LOD respectively loaded graphene nano-grid and layer structure. (b) and (c) are respective schematic diagrams of presumed detection mechanisms of grid and layer structure based graphene films.
The mechanism of this improvement can be illustrated as follows: in most electrochemistry reaction, the determinants are always concluded to the catalytic ability and electron transfer rate. Here the most different factor between two graphene structures is the environment of enzyme immobilization. Moreover, in grid structure, enzyme can be encompassed by the sphere graphene wall. On the contrary, normal layered shape can only touch the bottom protein which causes the waste of contact area between graphene and enzyme, and increase of transfer resistance. Besides, huge amount enzyme can be uniformly allocated to avoid the partial over-aggregation to produce an enhancement of enzyme reaction area. Above features can not only benefit the biosensor fabrication, but also the related protein reactions. Moreover, an extra advantage is occurred due to the material property of graphene. Graphene has been confirmed that it owns an ability of active center (flavin adenine dinucletide, FAD) connection of enzyme (Kulia et al, 2011; Yang et al, 2010; Zhang et al, 2014). Therefore, as shown in Fig. 6 (b) and (c), the special surrounding style of graphene 3D morphology can provide more electron channels to connect protein FAD and electrode surface. Obviously, the electrochemical signals of enzyme reaction can be much faster and stronger.
4. Conclusions A 3D nano-grid of thiol grafted graphene film has been in-situ constructed on Au surface. Relied on the function of thiol group, proteins can be uniformly captured into the cavities of nano-grid to serve as a homogeneous distributor. The efficiency of this distributor will not be constricted by the category and amount of proteins. The graphene distributor based biosensors have been confirmed to obtain a three times enhancement of performance due to the increase of reaction area and electron transfer channels. We believe that this powerful and general distributor will enrich protein immobilization methods to arouse a significant advance in performance, and the design of graphene mico-structure can be also promising in the future exploitation in the fabrications of functional graphene materials and their intriguing applications.
ACKNOWLEDGMENT
This work was supported by the Innovative Research Team Program by the Ministry of Education of China (No. IRT13070), Doctoral Fund of Ministry of Education of China (20113221110001), the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions (No. IRT13070), and the National Natural Science Foundation of China (No. 21176115).
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Figure Captions Figure 1
The synthesis process scheme of graphene nano-grid based distributor.
Figure 2
(a)The frequency and mass change of quartz crystal in graphene deposition process by QCM monitor; (b) to (d) is the structure evolution of graphene film surface respectively prepared under 200 s, 400 s and 600 s. (e) is the image under low magnification rate of (d); (g) to (h) are the HRTEM images of half-filled PS balls.
Figure 3
Nano-indentation test of prepared graphene film: (a) is the diagram of applied indenting force; (b) is the digital photo of indention surface; (c) is the FESEM image of indented region of film surface.
Figure 4 FESEM images of GOD loaded (a) graphene nano-grid (b) graphene layered film. The bar of inset is 1 m. (c) and (d) are LOD and BSA loaded graphene nano-grid, respectively. Figure 5 CV comparisons of graphene nano-grid based (a) glucose biosensor and (b) lactate biosensor before and after the respectively additions of 2 mM glucose and lactate Figure 6 (a) The fitted diagram of GOD and LOD respectively loaded graphene nano-grid and layer structure. (b) and (c) are respective schematic diagrams of presumed detection mechanisms of grid and layer structure based graphene films. Highlights z z z z
A single layered nano-grid structure of graphene was first synthesized. This special film can uniformly distribute proteins in its homogenous cavities. Various proteins can all be appropriately distributed with different categories and amount. Sensitivities of prepared biosensors can enhance three times than random enzyme immobilization.
3D graphene nano-grid as a homogeneous protein distributor for Ultrasensitive biosensors Zhenyu Chu, Lei Shi, Wanqin Jin
www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(14)00338-8 http://dx.doi.org/10.1016/j.bios.2014.05.006 BIOS6767
To appear in:
Biosensors and Bioelectronics
Received date: 10 March 2014 Revised date: 28 April 2014 Accepted date: 2 May 2014 Cite this article as: Zhenyu Chu, Lei Shi, Wanqin Jin, 3D graphene nano-grid as a homogeneous protein distributor for Ultrasensitive biosensors, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2014.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
3D graphene nano-grid as a homogeneous protein distributor for ultrasensitive biosensors Zhenyu Chu, Lei Shi and Wanqin Jin* State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical engineering, Nanjing Tech University, 5 Xinmofan Road, Nanjing 210009, P. R. China. Corresponding Author * E-mail: [email protected]. Fax: +86 25-8317-2292; Tel: +86 25-8317-2266. Abstract: In order to realize the protein uniform immobilization, a 3D nano-gird architecture of thiol grafted graphene film was fabricated to serve as a novel linker between protein and substrate. Relied on the online monitor by QCM, graphene deposition process can be exactly controlled to construct the perfect and continuous cavities with the consistent size of 500 nm. The synergetic characterization of FESEM and Nano-indentation characterizations have revealed the strong stability of grid structure to provide a firm foundation for further protein adsorption. Instead of common partial aggregation behavior, proteins can be spontaneously distributed into cavities by the interaction from thiol group. According to the verifications of various proteins, the efficiency of this distributor will not be constricted by the category and amount of protein, which exhibit its versatility of homogeneous distribution. Glucose and lactate oxidase loaded graphene distributors were directly served as biosensors to verify the superiority of distribution. Their sensitivities can be remarkably improved three times since the adoption of this nano-grid structured graphene distributor.
Keywords: Graphene film; In-situ synthesis; 3D nano-grid; Various proteins immobilization; Ultrasensitive biosensors 1. INTRODUCTION Generally, protein immobilization is an essential stage referred to the bio-materials fabrication (Chen et al, 2001), fermentation process design (Doran et al, 1986) and biosensor development (Mirsky et al, 1997). The immobilization status of protein, such as morphology, distribution and accumulation style, will synergistically affect the performance of bio-reaction process (Hudson et al, 2008; Onoda et al, 2012). Normally, the combination between protein and support which produces a weak interaction on interface is depended on a cross-linking reagent to produce a bridge function (Zhang et al, 2007; Niemeyer et al, 2001; Delamarche et al, 1997). It is traditionally consisted by the high viscous polymer to serve as a “glue” for protein adhesion. Hence, the control of protein distribution is difficult due to the rarely uniform coverage of cross-linker on substrate surface. In order to realize the regular distribution, only relying on the uncontrollable “glue” is useless. Therefore, a novel linker which is required to be of excellent biocompatibility, thermal stability and mass transfer ability should be introduced to build a strong chemical interaction of interface between protein and substrate. Besides, with the purpose of regular protein distribution, the structure of this linking layer should also possess a uniform microstructure to induce the protein settling down. Graphene has aroused the numerous attentions of various scientific fields because of its abundant unique properties. Its high surface area, rapid conductive rate and good environment stability are most encouraged to serve as superior advantages in bio-material fabrication (Patil et al, 2009; Song et al, 2010; Dong et al, 2011). However, because of the sole sheet shape, graphene morphology of formed film most keeps the layered accumulation. More structures of graphene films are difficult to be created. According to recent literatures, three dimensional (3D) graphene films which were
normally prepared by the reduction of graphene oxide have been confirmed to own higher performance due to the larger contact area and easy device construction (Hu et al, 2013; Dong et al, 2012). But the cavity size and location of these structures are still random formed which cannot provide a uniform and proper space for protein loading. The uniform distribution of protein can effectively avoid the partial aggregation behavior which is benefited to obviously increase the contact area and reduce the mass transfer resistance. Therefore if a homogeneous distribution to protein is expected to realize, the key is realization of a highly uniform structure of graphene for the protein immobilization.
Figure 1. The synthesis process scheme of graphene nano-grid based distributor.
In this work, thiol grafted graphene was adopted as a linking substance to produce a bridge function between protein and substrate. Relied on an online monitor strategy, the graphene layer was accurately shaped to a 3D nano-grid structure with a numerous 500 nm cavities by nanolithography approaches. Various proteins, glucose oxidase, lactate oxidase and bovine serum albumin, were respectively tested to verify the versatility of this protein uniform distributor. Based on the chemical interactions of thiol group belonged to graphene, proteins can be not only stably adhered on metal surface, but also uniformly and regularly distributed into the cavities of graphene grid. Moreover, glucose and lactate oxidase loaded protein distributors have be directly served as highly sensitive biosensors to exhibit the advance features of homogeneous protein distributors.
2. EXPERIMETAL 2.1 Reagents and apparatus Thiol graphene (4.0 wt% hydroxyl ratio) was synthesized by Jcnano technology Co., Polystyrene latex microspheres (PS, 500 nm, 2.5 wt% in water) were purchased from Alfa-Aesar
company.
K4[Fe(CN)6]·3H2O
(Sigma-Aldrich),
FeCl3·6H2O
(Sigma-Aldrich), (3-glycidyloxypropyl) trimethoxysilane (Sigma-Aldrich), sodium n-dodecyl sulfate (Alfa-Aesar), sodium L-lactate (Alfa-Aesar), glucose (Sinopharm Chemical Reagent Co., Ltd, China) and 30 wt% H2O2 (Sinopharm Chemical Reagent Co., Ltd, China) were of analytical grade purity and used without further purification. 4.5 mg glucose oxidase (GOD, EC 1.1.3.4, 168800 units per g, from Aspergillus niger, Sigma-Aldrich) and Bovine Serum Albumin (BSA, Sigma-Aldrich) were respectively dispersed in 1500 μL of 0.05 phosphate buffer solution with 0.1 M KCl (PBS, pH=6.5) and was sufficient for preparation of three electrodes. 2.5 mg lactate oxidase (LOD, EC 1.13.12.4, 20 units per mg, from Pediococcus sp., Sigma-Aldrich) was dispersed in 500 μL PBS and separated into 25 separate vials (2 units per 20 μL). All solutions were prepared with deionized water. Electrochemical characterizations were realized by an electrochemical workstation (CHI 660C, Shanghai Chenhua, China). All cyclic voltammetry (CV) experiments were operated in PBS at 25℃. A Pt wire and Ag/AgCl were used as the counter and reference electrodes, respectively. The scan rate was 50 mV s-1. Electrochemical Impedance Spectroscopy (EIS) characterization was carried out at the -0.05 V potential. The measurement of the voltage amplitude of 5 mV from the frequency range 0.05 Hz to 105 Hz was made in the buffer solution. Online monitoring of graphene coverage was fulfilled by quartz crystal microbalance (QCM200, Stanford Research Systems, Inc, USA). All field emission scanning electron microscopy images (FESEM) were taken by Hitachi-4800. The mico-structure of graphene was detected by high-resolution transmission electron microscopy (HRTEM) (JEM-2010 UHR, Japan). Prior to the
HRTEM measurements, the graphene film was scraped from the electrode surface, ultrasonically dispersed in ethanol. Nano-indentation characterization was operated on NanoTest Vantage (Micro Materials, UK). 2.2 Pretreatment of the Au disk In order to satisfy the clean and plane demands to substrate, Au disk (2 mm diameter) was first polished as a mirror by metallographical sand paper. Then the disk was dipped into a Piranha solution (7:3 mixture of H2SO4 and H2O2, v/v) for 30 min and rinsed with water. Finally, it was washed in ultrasonic environment for 30 min for ready usage. 2.3 Preparation of graphene nano-grid Since finishing above pretreatment step, one drop of PS beads was firstly added on Au disk which is enough to cover whole metal surface. The disk was held stationary for 1 min to enable sufficient combination of PS and Au. Secondly, the substrate was slowly immersed into deionized water, and then one or two droplets of 2% Sodium n-dodecyl sulfate were dropped into this water to change the surface tension which is aimed to disperse PS to form monolayer. Subsequently, the prepared monolayer of PS modified electrode was dried at 80 ˚C for 90 min in order to fix the beads to the surface. 5 mg thiol graphene powder was added into 20 ml ethanol to form graphene deposition source. In order to smash the graphene sheet to smaller size, this suspension liquid was then dispersed and vibrated by a probe-type sonicator (KBS-650, Kunshan Shumei, China) with a 20 KHZ of ultrasonic frequency for 30 min. Subsequently, the prepared monolayer PS/Au disk was vertically suspended to immerse the graphene source. During the deposition process, the liquid temperature was kept to 60 ˚C with stir in order to continuously condense the graphene source. After above step, disk was washed to clean the surface, and then moved to toluene for 30 min. Finally, the disk was dried in oven at 60 ˚C for 30 min.
2.3 Immobilization of proteins GOD, LOD and BSA were respectively applied as samples of protein. Graphene/Au disk was immersed into the divided enzyme solution for 1 hour. Then the disk was washed clean and ready for usage. 3. RESULTS AND DISCUSSION 3.1 Online control of graphene nano-grid formation
Figure 2. (a)The frequency and mass change of quartz crystal in graphene deposition process by QCM monitor; (b) to (d) is the structure evolution of graphene film surface respectively prepared under 200 s, 400 s and 600 s. (e) is the image under low magnification rate of (d); (g) to (h) are the HRTEM images of half-filled PS balls.
In order to build a 3D structure in graphene fabrication, as shown in Fig. 1, three steps are required to exactly fulfil. First step of template layout must ensure the regular and tight distribution of each PS nano-sphere. Subsequent process, graphene adsorption, is a key to the success of morphology construction. Amount of bonded graphene on Au surface will directly affect the integrity of final formed grid architecture. In this case, the accurate judgment of time node during graphene deposition is crucial. Hence, QCM technique was applied as an online strategy for the monitor of graphene adsorption
evolution. The whole graphene deposition process was detected by QCM equipment. The data of graphene amount shift on Au surface was online recorded. As shown in Fig. 2(a), total deposition process has happened two obvious adsorption stages: time periods before and after 500 s. Neglection of beginning instable process before ca. 100 s, the amount of graphene occurred a slow climb which indicated the initial combination of graphene on Au surface by strong Au-SH bond. It should be noticed that the space created by the neighbouring spheres is so narrow that only tiny graphene sheet can pass to arrive on metal surface for initial adsorption. Then, at the time of 500 s, a sudden elevation occurred to illustrate a rapid adsorption behaviour of graphene. This is due to the concentrating deposition method we adopted. Within 60 ˚C ethanol environment, graphene will continuously undergo a concentration to accelerate the “wall” building to surround the spheres on already formed foundation. Subsequently, the deposition again reached a stable stage which belongs to a steady aggregation between graphene sheets. Further deposition will cause the over-thickness of graphene film to cover whole template surface (time of 800 s, S1) which affects the grid formation. Accordingly, the whole preparation of graphene around template can be considered termination beyond ca. 600 s. For directly confirming the morphology evolution, the template removed graphene disks prepared by different deposition time were characterized by FESEM. The deposition periods of 200, 400, 600 s were selected as the typical samples. Exhibited in Fig. 2 (b), initially, the first arrival graphene sheets were crossed through the space between each PS sphere to combine with Au support. This amount of graphene is low to only surround the very bottom of PS, hence, since the removal of PS, just leaving a thin film with round traces. With prolonging the adsorption time, the graphene “wall” was bricked by graphene accumulation. According to the side view image in Fig. S2, it shows that graphene has covered the bottom region and partial gaps between PS balls. However, it is obvious that there are still some deficiencies to reveal in Fig. 2(c). Since
reaching a half-filled stage, we finally obtained an integrated and smooth grid of single layer structure with regular and uniform 3D nano-cavities of ca. 500 nm (Fig. 2(d)). Moreover, instead of just small region, a very large area of support can be covered with perfect nano-grid graphene film in Fig. 2(e). With the purpose to investigate the growth mechanism of grid wall, the graphene accumulation behavior was magnified under a HRTEM observation. The film of graphene covered PS spheres was peeled from Au surface, and dispersed in ethanol solution for TEM characterization. Based on the results in Fig. 2(f) to (h), it can be illustrated that multiple layers of films were covered on the sphere surface. Each layer may be not the same length, but the total length which relied on the stack is still long and continuous without fault. Further focusing on each layer, the thickness is ca. 3.4 Å which matches the standard graphene parameter (Ohta et al, 2006). Above evidences can indicate that, after the initial base film formation, the construction of grid wall structure is built by the encompassment of graphene sheets on the surfaces of PS beads. Then, the PS template was removed to leave the cavities and connected graphene walls to exhibit grid morphology. CV characterization measurement was applied to investigate the catalysis and electron transfer abilities of graphene nano-grid (Fig. S3). Bare Au disk, PS template covered Au (PS/Au), graphene modified PS/Au and graphene nano-grid modified Au (GE/Au) were respectively characterized by CV in the 0.05 M [Fe(CN)6]4-/[Fe(CN)6]3solution. With the calculation of each CV curves, graphene nano-grid is of the lowest resistance. Accordingly, the formation of nano-grid structure owns the best performance of reaction rate and resistance. This is attributed to the regular construction of film which can provide a uniform environment and channel to arouse the advantages of graphene in catalysis and electron transport. EIS technique was also applied to obtain the resistance data before and after the grid formation. According to the simulation of scanning data by equivalent circuit, the interface resistance between graphene and Au were calculated as 462.4 and 1291.2 Ω before and after PS template removal. This result
is consistent with above potential difference from CV characterization. It is mainly due to the existence of PS which owns low conductivity among graphene layers. Since the reaction beginning, the transfer channel between graphene and Au will be occupied by a layer of PS beads to slow down the amount of delivered electron which weaken the received current signal. 3.2 Protein distribution by graphene nano-grid
Figure 3 Nano-indentation test of prepared graphene film: (a) is the diagram of applied indenting force; (b) is the digital photo of indention surface; (c) is the FESEM image of indented region of film surface.
Before the immobilization of protein, the stability of graphene grid should be tested to provide a reliable environment for adsorption. A common trouble on graphene film is that it will often roll to change the morphology without a firm force to be adhered on support surface (Yu et al, 2007). This is a big challenge to maintain the stability of protein immobilization and application. Therefore, a nano-indentation measurement was adopted to investigate the interaction between Au substrate and graphene film. One prepared graphene grid film was scratched by an increasing force (Fig. 3(a)) from a diamond probe. As shown in Fig. 3(b), the trace of ravine was deeper and more obvious with the strength enhancement till the film had been thoroughly broken into two parts. We selected the tip scratch where began to happen the crack to characterize the micro-morphology of film. According to the surface image in Fig. 3(c), it can be found that, except the crack, the continuity and integrity of film had been maintained. Holes
also kept its original shape and size instead of deformation caused by the loss of constraint. Besides, the fracture surface did not roll up which illustrated that the elastic deformation was conquered by the substrate binding force of Au and –SH group. Above evidences can demonstrate that the graphene film can preserve its perfect grid and hole structure to resist the external force by the firm interaction between film and substrate. Subsequently, the proteins loading efficiency of graphene nano-grid was checked. In order to testify the versatile function in protein distributions, three different proteins: GOD, LOD and BSA were served as samples. GOD and LOD both belong to the oxidase category, but loading amounts are very different in real application. Different with oxidase, BSA is one of serum albumin. Therefore, the capacities of graphene grid on amount and category can be tested. In the whole immobilization process, proteins kept free adsorption without other external forces or cross-linkers. As our expectation, proteins will be captured into the cavities by the attachment function of thiol groups from graphene wall. Hence, the bonding behaviors between enzyme and graphene are prerequisite. The characteristic group changes of graphene nano-grid which was loaded by GOD were characterized by FT-IR in Fig. S5. Two extra peaks generate in IR curve, one locates at 1543 cm-1 and another places at 1633 cm-1. They are the representative groups: amide I and amide II which is general existence in protein structure (Wu et al, 2010). In the IR result of only graphene nano-grid, at 804 cm-1, there is a weak peak from C-SH bending (Pham et al, 2013). But it has disappeared since GOD immobilization. This illustrates free C-SH group has participated in the reaction of protein loading. This combination is a typical reaction between protein and –SH grafted material which is depended on the redox production of –S-S– group from protein and –SH group modified substance (Wilkinson et al, 2004; Yiu et al, 2001; Ovsejevi et al, 2009). Hence, around the thiol graphene formed grid architecture, proteins are promising to be arranged along hemispherical shape.
Figure 4. FESEM images of GOD loaded (a) graphene nano-grid (b) graphene layered film. The bar of inset is 1μm. (c) and (d) are LOD and BSA loaded graphene nano-grid, respectively.
GOD is one of the most applied proteins in biological detection. In order to harvest a high performance, its distribution is normally required as uniform as possible. Here, as shown in Fig. 4(a), the whole GOD loaded film surface was extremely uniform. The profile of graphene grid was still clear to be distinguishable. According to the magnified image of inset, the space of original cavities was not only absolutely filled by GOD, but also formed continuously round bulges with 500 nm size. On the contrary, if grid structure was not adopted, protein would aggregate together without restriction, and this layer would be consisted by some separated, irregular and uneven GOD clusters with much larger size (Fig. 4(b)). Meanwhile, LOD which was only 1/15 amount of GOD in real usage was repeated to load on graphene surface. FESEM characterization indicated that except some unfilled cavities due to the fewer amounts, graphene grid can still distribute protein uniformly along its framework. Besides of enzyme, another category of protein, BSA, was also attempted. Under the same loading amount as GOD, the distribution behavior and final surface morphology were nearly consistent. Consequently, the fabricated graphene nano-grid can be of a generally homogeneous
distribution function to the protein immobilization. 3.3 Effects of protein homogeneous distributions
Figure 5. CV comparisons of graphene nano-grid based (a) glucose biosensor and (b) lactate biosensor before and after the respectively additions of 2 mM glucose and lactate
The superiority of graphene based enzyme biosensor is the direct electron channel between electrode and active center of enzyme (flavin adenine dinucletide, FAD) instead of the necessity of mediators in traditional fabrication (Shao et al, 2010). Therefore, the operation potential of graphene based biosensor is usually much lower. As shown in Fig. 5, since the addition of 2 mM target glucose into the detection system, an obvious signal increase happened at the potential of ca. -0.6 V. The same phenomenon was also occurred in the lactate detection. Above results illustrate that prepared graphene film owns a strong response to the oxidase reaction at a special potential, and the reacted electrons can fast transfer through graphene layer to electrode surface. Accordingly, in the following performance characterizations, the operation potentials were determined. The
effects
of
protein
homogeneous
distribution
were
studied
by
a
chronoamperometry measurement. Taking GOD and LOD as models, the bio-sensing performance of nano-grid structure was compared with layer structure (the film morphology
was
shown
in
Fig.
4(b)).
According
to
the
calculation
of
chronoamperometry data (Fig. 6(a)), for GOD loading, homogeneous distribution can reach a sensitivity of 35.06 μA·mM·cm-2, and random adsorption was only 5.12 μA·mM·cm-2. Similarly, LOD loaded grid architecture also exhibited a nearly four times exceedance than layered distribution (9.98 vs. 2.76 μA·mM·cm-2). The relative standard deviation (RSD) of prepared graphene nano-grid biosensor is 10.1%. The reusability of prepared graphene grid based electrode was also investigated. The same electrode which was used by chronoamperometry characterization was stored in fridge at 4 ℃, and then repeated to test after 10 days. The sensitivity can only decreased to 87% which illustrates the excellent reusability of grid film. This may be attributed to the strong interface interactions of enzyme/graphene and graphene/Au by –SH group. In order to realize
the
quantitative
evaluation
of
enzyme
immobilization
behavior,
a
Michaelis-Menten constant KM in enzyme kinetics was calculated. It can represent the substrate concentration at which the reaction rate is at half-maximum, and is an inverse measure of the substrate's affinity for the enzyme (Kamln et al, 1980). The calculated KM for graphene grid based glucose biosensor is 0.71 mM. Compared with other graphene based glucose biosensors in Tab. S1, the KM constant, sensitivity and operation potential are all superior to only graphene film, even better than other high catalytic material doped graphene films. This excellent performance is due to the uniform distribution of enzyme immobilization. It will avoid the instability of enzyme caused by random accumulation on partial sites, and also increase the contact area of enzyme for target to obviously enhance the response signal in detection. Moreover, due to the low application potential, as-prepared biosensor can own an outstanding anti-interference ability in the co-existence of ascorbic acid and uric acid (Fig. S7). Therefore, the homogeneous protein distribution brings a superior behavior of performance.
Figure 6. (a) The fitted diagram of GOD and LOD respectively loaded graphene nano-grid and layer structure. (b) and (c) are respective schematic diagrams of presumed detection mechanisms of grid and layer structure based graphene films.
The mechanism of this improvement can be illustrated as follows: in most electrochemistry reaction, the determinants are always concluded to the catalytic ability and electron transfer rate. Here the most different factor between two graphene structures is the environment of enzyme immobilization. Moreover, in grid structure, enzyme can be encompassed by the sphere graphene wall. On the contrary, normal layered shape can only touch the bottom protein which causes the waste of contact area between graphene and enzyme, and increase of transfer resistance. Besides, huge amount enzyme can be uniformly allocated to avoid the partial over-aggregation to produce an enhancement of enzyme reaction area. Above features can not only benefit the biosensor fabrication, but also the related protein reactions. Moreover, an extra advantage is occurred due to the material property of graphene. Graphene has been confirmed that it owns an ability of active center (flavin adenine dinucletide, FAD) connection of enzyme (Kulia et al, 2011; Yang et al, 2010; Zhang et al, 2014). Therefore, as shown in Fig. 6 (b) and (c), the special surrounding style of graphene 3D morphology can provide more electron channels to connect protein FAD and electrode surface. Obviously, the electrochemical signals of enzyme reaction can be much faster and stronger.
4. Conclusions A 3D nano-grid of thiol grafted graphene film has been in-situ constructed on Au surface. Relied on the function of thiol group, proteins can be uniformly captured into the cavities of nano-grid to serve as a homogeneous distributor. The efficiency of this distributor will not be constricted by the category and amount of proteins. The graphene distributor based biosensors have been confirmed to obtain a three times enhancement of performance due to the increase of reaction area and electron transfer channels. We believe that this powerful and general distributor will enrich protein immobilization methods to arouse a significant advance in performance, and the design of graphene mico-structure can be also promising in the future exploitation in the fabrications of functional graphene materials and their intriguing applications.
ACKNOWLEDGMENT
This work was supported by the Innovative Research Team Program by the Ministry of Education of China (No. IRT13070), Doctoral Fund of Ministry of Education of China (20113221110001), the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions (No. IRT13070), and the National Natural Science Foundation of China (No. 21176115).
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Figure Captions Figure 1
The synthesis process scheme of graphene nano-grid based distributor.
Figure 2
(a)The frequency and mass change of quartz crystal in graphene deposition process by QCM monitor; (b) to (d) is the structure evolution of graphene film surface respectively prepared under 200 s, 400 s and 600 s. (e) is the image under low magnification rate of (d); (g) to (h) are the HRTEM images of half-filled PS balls.
Figure 3
Nano-indentation test of prepared graphene film: (a) is the diagram of applied indenting force; (b) is the digital photo of indention surface; (c) is the FESEM image of indented region of film surface.
Figure 4 FESEM images of GOD loaded (a) graphene nano-grid (b) graphene layered film. The bar of inset is 1 m. (c) and (d) are LOD and BSA loaded graphene nano-grid, respectively. Figure 5 CV comparisons of graphene nano-grid based (a) glucose biosensor and (b) lactate biosensor before and after the respectively additions of 2 mM glucose and lactate Figure 6 (a) The fitted diagram of GOD and LOD respectively loaded graphene nano-grid and layer structure. (b) and (c) are respective schematic diagrams of presumed detection mechanisms of grid and layer structure based graphene films. Highlights z z z z
A single layered nano-grid structure of graphene was first synthesized. This special film can uniformly distribute proteins in its homogenous cavities. Various proteins can all be appropriately distributed with different categories and amount. Sensitivities of prepared biosensors can enhance three times than random enzyme immobilization.
