Enzyme-Encapsulated Liposome-Linked Immunosorbent Assay

Subscriber access provided by University of Massachusetts Amherst Libraries

Article

Enzyme Encapsulated Liposome-Linked Immunosorbent Assay Enabling Sensitive Personal Glucose Meter Readout for Portable Detection of Disease Biomarkers Bingqian Lin, Dan Liu, Jinmao Yan, Zhi Qiao, Yunxin Zhong, Jia-Wei Yan, Zhi Zhu, Tianhai Ji, and Chaoyong James Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00777 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on February 29, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Enzyme Encapsulated Liposome-Linked Immunosorbent Assay Enabling Sensitive Personal Glucose Meter Readout for Portable Detection of Disease Biomarkers Bingqian Lin, Dan Liu, Jinmao Yan, Zhi Qiao, Yunxin Zhong, Jiawei Yan, Zhi Zhu, Tianhai Ji, Chaoyong James Yang*

State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Affiliated Chenggong Hospital, Xiamen University, Xiamen 361005, P. R. China. * To whom correspondence should be addressed. Tel: (+86) 592-218-7601; Fax: (+86) 592-2189959. E-mail: [email protected]

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 27

ABSTRACT:

There is considerable demand for sensitive, selective and portable detection of disease-associated proteins, particularly in clinical practice and diagnostic applications. Portable devices are highly desired for detection of disease biomarkers in daily life due to the advantages of being simple, rapid, user-friendly and low-cost. Herein we report an enzyme-encapsulated liposome-linked immunosorbent assay for sensitive detection of proteins using personal glucose meters (PGM) for portable quantitative readout. Liposomes encapsulating a large amount of amyloglucosidase or invertase are surface-coated with recognition elements such as aptamers or antibodies for target recognition. By translating molecular recognition signal into a large amount of glucose with the encapsulated enzyme, disease biomarkers such as thrombin or C-reactive protein (CRP) can be quantitatively detected by a PGM with high a detection limit of 1.8 nM or 0.30 nM, respectively. With the advantages of portability, ease of use, and low-cost, the method reported here has potential for portable and quantitative detection of various targets for different POC testing scenarios, such as rapid diagnosis in clinic offices, health monitoring at the bedside, and chemical/biochemical safety control in the field.

KEYWORDS: Enzyme encapsulated liposome, personal glucose meter, portable detection, disease biomarkers, signal amplification


2

Page 3 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Point of care testing (POCT) is a rapidly developing field involving near-patient, bedside, ancillary, or in-field testing.1 It enables a wide range of targets to be detected easily and quickly without the use of sophisticated equipment or well-trained professionals.2 POCT is important for the developing world or resource-limited areas in diagnostics, environmental monitoring, as well as food quality testing.3 In the last few years, considerable effort has been devoted to develop patient self-testing devices to detect pregnancy, blood pressure, drugs of abuse, blood glucose, etc.4 The personal glucose meter (PGM) is one of the most successful examples of patient selftesting. These devices have been widely applied in the household scenarios due to their compact size, low price, reliable quantitative results and easy operation.5 Traditionally PGMs have been used only for the detection of glucose in blood. To make full use of the portable and userfriendly PGMs for non-glucose targets, Lu’s group reported a pioneer work using PGMs to detect drugs, biological cofactors, disease markers, toxic metal ions6-9 and nicotinamide coenzymes10 by using aptamer conjugated invertase to convert aptamer/target recognition into glucose release. This concept has been further extended for the detection of different kinds of targets. For example, Xu and co-workers developed a method combining magnetic beads and DNA to detect mercury (II) ion using PGM with a detection limit of 8 nM.11 Jeon’s group reported a method using antibody-functionalized magnetic nanoparticle clusters for the detection of Salmonella bacteria in milk using PGM with a limit of 10 cfu/mL.12 Wang’s group developed a method combining a microfluidic chip and PGM to detect nucleic acids to achieve point-ofcare detection.13 They also reported a method to detect cardiac biomarker myoglobin using aptamer and PGM.14 Other enzymes were also used to combine PGM to achieve the detection of


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 27

non-glucose targets. Lu and his coworker using PGM and alkaline phosphatase for portable detection of galactose-1-phosphate uridyltransferase in clinical galactosemia diagnosis.15 Wang’s group used maltopentaose and α-glucosidase to achieve one-step quantitative detection of αamylase in serum and urine.16 The above methods used invertase to convert target recognition into glucose release, which requires chemical conjugation of invertase with an antibody or aptamer through multiple-step and time-consuming chemical reactions. Another potential limitation of chemical conjugation loss of enzyme function. To avoid the need for chemical conjugation of target recognition element with glucose producing enzymes, we have reported a target-responsive aptamercrosslinked “sweet” DNA hydrogel with entrapped amylase to detect non-glucose targets with high sensitivity and accuracy.17 Upon interaction with the target molecule, the hydrogel dissolves because of aptamer/target binding leads to breakage of the crosslinker of the linear polymer chains. As a result, amylase is released from the hydrogel to catalyze the hydrolysis of amylose to generate a high concentration of glucose for sensitive readout. The DNA-hydrogel integrates the molecular recognition element with enzyme using reversible physical trapping, thus avoiding the need for chemical coupling antibody or aptamer with enzyme. Highly sensitive and selective detection of several non-glucose targets such as cocaine and ATP have been demonstrated.17 The target-responsive aptamer-crosslinked “sweet” DNA hydrogel approach is potentially useful for the detection of a variety of non-glucose targets using PGMs by replacing the aptamer with a target-related sequence. Unfortunately, the response time of the DNA hydrogels depends heavily on the molecular size of the target.18 The DNA hydrogel responds quickly to small molecules, but it shows slow response to large targets because of the slow diffusion time. Thus, a long reaction time is required for large molecules such as disease biomarker proteins, and this is


4

Page 5 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


not desirable for POCT. Therefore, it is important to develop new strategies to convert a molecular recognition event into a glucose-producing reaction to enable rapid, simple, sensitive and portable detection of large biomolecules using a PGM. Liposomes are spherical structures composed of a bilayer of phospholipids or any similar amphipathic lipids with an aqueous phase inside.19 Due to their large internal volume, molecules can be encapsulated within the aqueous inner cavity or trapped within the lipid tails of the bilayer.20,

21

For analytical sciences, liposomes can be functional components for signal

amplification by encapsulating a large number of signal molecules which may be released under controllable conditions.21-25 Furthermore, the surface of liposomes can be easily functionalized with different molecular recognition elements such as antibodies and aptamers. More importantly, proteins and small molecules can be encapsulated as signal output medium without the need for chemical modification. Herein, we present a general enzyme-encapsulated liposome-linked immunosorbent assay method for portable detection of proteins using PGMs. In this method, liposomes are used to non-covalently entrap a large amount of amyloglucosidase or invertase for signal transduction and signal amplification. When used as a reporter element in the ELISA format, the liposome surface-coated with recognition elements such as aptamers or antibodies can be controllably dissociated to release amyloglucosidase to catalyze the glucose-producing reaction for final sensitive readout by a PGM. Using the enzyme-liposome method, disease biomarkers such as thrombin and C-reactive protein (CRP) can be quantitative detected by a PGM with high sensitivity. With the advantages of portability, ease of use, and low-cost, the method reported here can be applied for easy and reliable detection of a wide range of targets for different POC


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 27

testing scenarios, such as rapid diagnosis in clinic offices, bedside health monitoring, and chemical/biochemical safety control in the field. Materials and methods Materials and Reagents. Streptavidin-modified magnetic beads (MBs, 1 µm in diameter) were purchased from Invitrogen Inc. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from TCI Inc. Cholesterol was obtained from Sigma-Aldrich, Inc. 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene

glycol)-2000]

(DSPE-PEG)

and

extruder components were purchased from Avanti Polar Lipids, Inc. Amyloglucosidase from aspergillus niger and Grade VII invertase from baker’s yeast were purchased from SigmaAldrich, Inc. Thrombin was obtained from Haematologic Technologies, Inc. Human serum albumin (HSA) and Goat Anti-Rabbit IgG (IgG) were purchased from Abcam Inc., Lysozyme (Lyso) was purchased from R&D System. Recombinant human C-reactive protein (CRP), Mouse monoclonal anti-human CRP antibody (MAB17071) and biotinylated mouse IgG (BAM17072) were from R&D System. Biotin N-hydroxysuccinimide ester (BNHS) was purchased from Sigma-Aldrich, Inc. All reagents were analytical grade and used as received. Millipore ultrapure water (18.2 MΩcm) was used throughout the experiment. The commercial personal glucose meter used in this study was an Ake Ling Rui blood glucose meter. Synthesis of oligonucleotides. The DNA sequences used for this work were synthesized on an ABI 394 DNA Synthesizer based on standard DNA synthesis protocol. The sequences of aptamers are shown in the supporting information. A 15-mer aptamer against thrombin (Capture probe) was modified at the 3’-end using biotin labeled CPG. A 29-mer aptamer against thrombin (Detection probe) was modified at the 5’-end using the lipid phosphoramidite. After synthesis and modification, the DNA product was separated from the solid support, and deprotected with


6

Page 7 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ammonia treatment. Afterwards, DNA was purified with an LC3000 semi-preparative HPLC system using a C18 column for Capture probe and a C4 column for Detection probe using 0.1 M triethylamine acetate (pH 7.0)/acetonitrile as the mobile phase with a flow rate of 1 mL min-1. Preparation of enzyme encapsulated liposomes. Liposomes were prepared by the thin lipid film-hydrated method as described elsewhere with slight modifications.26 As shown in Scheme S2, a mixture containing DPPC (dipalmitoyl-sn-glycero-3-phosphocholine), cholesterol, and DSPE-PEG

(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene

glycol)-2000]) (100:50:5 molar ratio, 26 mg total) was first dissolved in 3 mL chloroform in a 100-mL round-bottom flask, then dried in a rotary evaporator to form a thin lipid film on the inside wall of the flask. The film was then hydrated in 1mL phosphate buffer (0.01 M, pH 7.4) containing 60 mg/mL amyloglucosidase or invertase at 35°C for 2 h to form liposomes. The liposomes were incubated in a 45°C water bath for 20 min before passing through the 0.4 µm polycarbonate film to produce a homogeneous suspension of liposomes with uniform size. The excess amyloglucosidase was removed by a dialysis bag with 300 kDa MWCO (Spectrum, USA) against 500 mL of PBS at 4°C with stirring for 48 h. During the dialysis, the PBS was changed several times. The average size of the liposomes were measured by using a dynamic light scattering system (Malvern Nano ZS90, USA). The AFM observations were performed with a Nano Scope II (Agilent Technologies, 5500AFM/SPM, USA). Before the AFM analysis, the solutions of liposomes were diluted in water in the portion of 1:100. Droplets of 40 µL sample were deposited on a small mica plate with a diameter of 1 cm. After 5 min, the remaining water was removed by paper filters, and the mica was drying with gentle nitrogen flow. Procedures for thrombin detection. Capture probe was conjugated on the magnetic beads (MBs) by means of streptavidin–biotin binding, and Detection probe was functionalized on the


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 27

surfaces of liposomes by inserting the lipid tail into the lipid bilayer via incubating the DNA and liposome for 30 min at 25°C. Compared to other methods to conjugate aptamer on nanomaterials for analytical applications27-29, using lipid tagged aptamer for directly insertion onto the surface of liposome is much easier and convenient. For thrombin detection, 40 µL 6 mg/mL MBs were incubated with different concentrations (from 0 to 250 nM) of human thrombin for 1 h at 25 °C (Tris 20 mM, NaCl 140 mM, KCl 5 mM, MgCl2 1 mM, pH 7.5). After washing with PBS buffer (pH 7.4) twice, 40 µL amyloglucosidase-encapsulated liposomes functionalized with Capture B was added to the MBs and incubated at 25°C for 1 h. After incubation, the MBs were washed with PBS buffer (pH 7.4) six times before adding them to 30 µL 3% amylose solution (1% Triton X-100, 0.1M PBS, pH 4.5 for the most suitable pH of amyloglucosidase) . After incubation for 1 h at 40 °C, 1 µL of the solution was removed for readout by a commercially available PGM (Ake Ling Rui, China). The whole analysis time is 3 h. Procedures for CRP detection. A 20-µL aliquot of 5 mg/mL CRP antibody-functionalized capture MBs was used. After removal of the supernatant by a magnet, the MBs were dispersed in 20 µL CRP sample solution of different concentrations from 0 ng/mL to 500 ng/mL and incubated for 30 min at 25°C (PBS buffer, 1% BSA, pH 7.4). Then, the MBs were washed three times (PBS buffer, 0.05% Tween-20, pH 7.4). After that, 20 µL of 8 µg/mL biotinylated detection antibody was added, followed by mixing at room temperature for 30 min (PBS buffer, 1% BSA, pH 7.4). The MBs were further washed three times (PBS buffer, 0.05% Tween-20, pH 7.4) before adding 20 µL of 8 µg/mL streptavidin in buffer (PBS buffer with 1% BSA, pH 7.4). Then the solution was incubated for 30 min. Later, the MBs were washed three times with PBS buffer (pH 7.4) and 20 µL of biotinylated liposomes containing encapsulated amyloglucosidase were added and incubated for 30 min at room temperature. Afterwards, the MBs were washed


8

Page 9 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


six times with PBS buffer. Finally, a 20 µL solution of 3% amylose (pH 4.5) and 4 µL TritonX100 were added and incubated at 50°C, and 1 µL mixture was tested by a PGM after 2 h. The whole analysis time is 4 h.

Results and Discussion Working principle of enzyme-encapsulated liposome-linked immunosorbent assay. The principle of the portable detection assay is shown in Scheme 1. Capture aptamer or antibody (Capture probe) functionalized magnetic beads are first introduced into the sample solution to capture the target protein. After brief washing, detection aptamer or antibody (Detection probe) functionalized liposomes are added. Capture probe and Detection probe bind with the protein to form a “sandwich” structure. After washing, amylose solution with Triton X-100 is added to break the liposomes leading to release of a large amount of amyloglucosidase encapsulated in the liposomes. The amylose can be catalyzed by amyloglucosidase to produce a large amount of glucose which can be quantitatively tested by the PGM. Considering the large cavity of the liposome for enzyme trapping and the high hydrolysis efficiency of amyloglucosidase, the liposome-based method can magnify the signal to a large extent, enabling highly sensitive detection of protein using a portable PGM.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 27

Scheme 1. Working principle of enzyme-encapsulated liposome-linked immunosorbent assay with beads / protein / liposome “sandwich” structure. Characterization of liposomes. Liposomes were prepared by the thin lipid film-hydrated method using DPPC, cholesterol, and DSPE-PEG in certain molar ratios and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). The liposomes were diluted in water (1:1000) for DLS analysis on a Malvern Instruments Zetasizer instrument. As shown in Figure 1A, the DLS results indicated that the liposomes have a uniform size of approximately 320 nm. Images of the liposomes were further obtained by AFM in the noncontact mode. The AFM image of liposomes is shown in Figure 1B. After deposition on the mica surface, liposomes showed a spherical form with slight deformation because the liposome was clinging to the mica with a short height of only 20 nm. The liposomes appeared as individual particles with the sizes of approximately 200-400 nm measured using the analysis software PicoView. The AFM result was consistent with the DLS value. The concentration of liposomes was calculated to be 4.5×1013/mL by dividing the total lipid molecule number by the number of lipid molecules per liposome. The number of lipid molecules


10

Page 11 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in one liposome was calculated as shown in eq (1) to be about 290000, as the bilayer thickness (T) was assumed as 4 nm.30 The radius (R) was calculated by the DLS result to be 160 nm. The average headgroup surface area per lipid molecule (A) was calculated from eq (2) to be 0.53 nm2/lipid, where A1, A2, A3 were 0.71 nm2, 0.19 nm2, and 0.41 nm2 for DPPC, cholesterol and DSPE-PEG, respectively.30 The p1, p2, p3 were the mole fraction of DPPC, cholesterol and DSPE-PEG, respectively, from the molar ratio of 100:50:5. N

(1)

A ∙ ∙ ∙ (2) The total concentration of amyloglucosidase in the liposome solution was determined to be 21 mg/mL by measuring the UV absorbance of a liposome solution of known concentration and comparing to a standard calibration curve of free amyloglucosidase. As a result, the number of amyloglucosidase molecules encapsulated in one liposome was 2896, which was large enough to magnify the signal of the target.

Figure 1. (A) DLS data indicating the uniform size of liposomes. (B) AFM image of liposomes. Rapid and controllable release of enzymes from liposomes. In the detection assay, the liposomes play a crucial role to achieve signal amplification and transduction. Slow release of enzymes from liposomes can prolong the detection time. More importantly, a rapid assay can


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 27

meet the need for point-of-care detection. Thus, it was of great importance to accomplish rapid and controllable release of enzymes from the liposomes. In this regard, nonionic surfactant TritonX-100 was used for rapid breakage of liposomes to release enzyme without compromising activity of the enzyme. To investigate the breakage kinetics of liposome, 1% Triton X-100 was added to a mixed solution containing 5 µL liposomes and 30 µL 3% amylose solution. The mixture was monitored by PGM every 2 minutes. As shown in Figure 2A, the liposomes rapidly released enzyme when the surfactant was added, and the signal reached the maximum measuring range of the PGM in 16 min, which was a suitable time for detection assay. The signal from three repeat experiments was reproducible as evidenced by a small relative standard deviation of less than 0.9%. In contrast, no response was observed when only PBS buffer was added. These results suggested that the liposome can be rapidly and controllably broken by addition of surfactant. Long term stability of liposomes. Liposomes can trap a large number of molecules to magnify the signal in the analytical assay. One of the important factors is the long term stability of liposomes. Long term stability can reduce the frequency of the preparation and keep the same signal level for further use in the detection assay. The long term stability of the amyloglucosidase encapsulated liposomes during storage was monitored through the hydrolysis of amylose by amyloglucosidase before and after lysis of the liposomes after storage at 4°C for 2 months. Triton X-100 (1%) was added to a solution containing 5 µL liposomes and 20 µL 3% amylose and was monitored after 5 minutes. As a control, PBS solution, instead of Triton X-100 was added. As shown in Figure 2B, the PGM signal slightly decreased (ca 20%) after storing the liposome for 2 months, In contrast, there was no response when only PBS buffer was added. The


12

Page 13 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


result indicated that the amyloglucosidase encapsulated liposomes were very stable with nearly zero leakage during storage.

Figure 2. (A) Kinetics of amyloglucosidase release from liposomes in response to the addition of surfactant. (B) Long-term stability characterization of liposomes at 0 day and 2 months. Enzyme-encapsulated liposome-linked immunosorbent assay for thrombin. Thrombin is a multifunctional serine protease which will present in blood and tissue in the situation of physiological and pathological blood coagulation.31 It is reported that when ischemia occurs, thrombin can be an endogenous mediator of hippocampal neuroprotection at low concentrations (50 pM to 100 nM) but causes degeneration at higher concentrations.31, 32 The liposome-based signal amplification method is designed for portable detection of thrombin using a glucometer. Different concentrations of thrombin ranging from 0 nM to 250 nM were tested in three parallel measurements. As shown in Figure 3A, the PGM signal increased linearly with the concentration of thrombin up to ∼250 nM, clearly indicating the quantitative detection capability of the liposome-based signal amplification method. The detection limit of this method was calculated to be 4.5 nM using 3σb/slope, in which σb was the standard deviation of control samples. The detection limit can be decreased to 1.8 nM by simply prolonging the reaction time of


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

amyloglucosidase with amylose from 1h to 1.5h. The sensitivity and dynamic range of the liposome-based method span the physiological concentration of thrombin from low nanomolar to low micromolar in resting and activated blood, respectively.33 In the liposome-based amplification method, the aptamer is embedded into the surface of the liposomes by hydrophobic interactions of the lipid tail conjugated to the aptamer rather than by covalent binding, which simplifies the procedure and allows easy control of the amount of aptamer. The method has an excellent versatility, as the enzyme encapsulated in liposomes can be changed to another glucose-producing enzyme such as invertase. To test this possibility, the invertase-encapsulated liposomes were prepared in the same way as those containing amyloglucosidase to quantify the concentration of thrombin. Different concentrations of thrombin from 0 nM to 50 nM were tested in three parallel measurements using invertaseencapsulated liposomes. As shown in Figure 3B, the PGM signal was also proportional to the concentration of thrombin, and the detection limit was calculated to be 1.7 nM .The value of the detection limit of the enzyme-encapsulated liposome-linked immunosorbent assay for thrombin with a glucometer readout is comparable to a limit of detection of 6.4 nM using electrochemical detection;33 10 nM using aptamer affinity chromatography with fluorescence detection;34 2.7 nM using a fluorescence method.35 It should be pointed out that these methods require large-scale instruments and professional operators, while the current method only requires an inexpensive, portable, user-friendly glucometer for signal readout. Comparing to the above methods, this assay showed great advantages of its easy-operation and portability, it was of great convenience in the resources-limited area. But it has to be pointed out clearly, that the PGM is limited to single analyte at the time.


14

Page 15 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (A) Detection of different concentrations of thrombin by the liposome-based signal amplification method using PGM with linear response range from 0 nM to 250 nM (black line). The sensitivity of the assay can be further increased by simply prolonging the time from 1 h to 1.5 h (red line). (B) Detection of different concentrations of thrombin by invertase-encapsulated liposomes using PGM with linear response range from 0 nM to 50 nM.

Selectivity of thrombin detection assay. The selectivity of the thrombin detection assay was demonstrated with several common proteins, such as lysozyme, human serum albumin (HSA) and goat-anti-rabbit IgG. The concentrations of these proteins were 1 µM and thrombin concentration was 100 nM. As shown in Figure 4A, negligible signal was observed upon addition of other proteins, while thrombin at 100 nM produced a high PGM signal. These results clearly show that the liposome-based signal amplification method can allow quantitative detection with PGM with high sensitivity and selectivity. Comparison with single-enzyme ELSA readout. To investigate the signal amplification effect of liposomes, the experiment of single amyloglucosidase ELISA was carried out. The biotinylated Detection probe and biotinylated amyloglucosidase conjugates were synthesized. In the presence of thrombin, the Capture probe and biotinylated Detection probe bind with thrombin


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

simultaneously to form a sandwich complex, and successive addition of streptavidin and biotin−amyloglucosidase conjugates makes it possible to bind biotin-amyloglucosidase conjugates to the beads. Thus, the biotin-amyloglucosidase conjugates can hydrolyze the amylose to produce glucose which can be detected by PGM. As shown in Figure 4B, the PGM signal of liposome-based method was almost 17-fold that of the single-amyloglucosidase ELISA even at a tenfold concentration of thrombin. This result clearly demonstrates that our liposomes, which encapsulate thousands of enzyme molecules, can be used for effective signal amplification.

Figure 4. (A) The selectivity of liposome-based signal amplification method. Concentration of thrombin was 100 nM, while other proteins were 1 µM. (B) Comparison of liposome-based signal amplification method with single amyloglucosidase. Detection concentration of liposomebased method was 200 nM, and the concentration of single amyloglucosidase method was 2000 nM. Detection of thrombin in a biologically complex sample. The ability of the liposome-based signal amplification method to detect the analyte in a biologically complex matrix was tested. Different concentrations of thrombin from 0 nM to 200 nM were spiked in 50% human blood


16

Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


serum. As shown in Figure 5, the PGM signal in the real sample was also proportional to the concentration of thrombin, and the detection limit was 12.8 nM. These results clearly indicate that the proposed liposome-based method has potential for detection of proteins in real biological samples.

Figure 5. Detection of different concentrations of thrombin by liposome-based signal amplification method using PGM in 50% serum with linear response range from 0 nM to 200 nM. Antibody-based enzyme-encapsulated liposome-linked immunosorbent assay. The liposome-based method can be applied in an aptamer sandwich assay as well as an antibody sandwich assay. To achieve the application of the method in an antibody sandwich assay, the ELISA experiment was carried out to detect CRP, which is an acute phase protein present in blood plasma.36 When an inflammatory process occurs, the concentration of CRP increases up to three orders of magnitude in the human body. It is reported that CRP concentration below 1 mg/L indicates low risk, between 1 and 3 mg/L indicates average risk, while 3 mg/L indicates high risk of cardiovascular diseases.37


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 27

In the CRP detection assay, the liposome was labeled with biotin via a DNA linker of 20 T embedded on the surface of liposomes by lipid modification. After antibody-conjugated MBs and biotin labeled secondary antibody bind with CRP to form a sandwich structure, streptavidin and biotin-labeled liposomes are added successively to form further structure. Finally, the amylose is added to the complex to produce glucose which can be detected by PGM. Different concentrations of CRP ranging from 0 ng/mL to 500 ng/mL were tested in three parallel measurements. As shown in Figure 6A, the PGM signal was proportional to the concentration of CRP up to ∼500 ng/mL, which indicated the detection capability of the antibody sandwich structure combined with liposome-based signal amplification. The detection limit of this method was calculated to be 35 ng/mL (0.30 nM), which completely meets the clinical demand to evaluate the risk level of cardiovascular disease from 1 mg/L to 3 mg/L. The selectivity of the CRP detection assay was tested with several common proteins, such as thrombin, human serum albumin (HSA) and goat-anti-rabbit IgG. The concentrations of these proteins were 2 µg/mL and the CRP concentration was 200 ng/mL. As shown in Figure 6B, negligible signal was observed upon addition of other proteins, while CRP at a concentration one order magnitude lower produced a high PGM signal.


18

Page 19 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (A) Detection of different concentrations of CRP by liposome-based signal amplification method using PGM with linear response ranging from 0 ng/mL to 500 ng/mL. (B) The selectivity of liposome-based signal amplification method. Concentration of CRP was 200 ng/mL, while other proteins were 2 µg/mL. To confirm the accuracy of our method, the quantitation results acquired by the liposomebased method for different concentrations of CRP were compared with the results of a traditional ELISA method. A positive correlation was found between the two methods (Figure S4), with a slope of 0.96 and a correlation coefficient of 0.94, suggesting that the accuracy and reliability of the liposome-based method is as good as that of the traditional ELISA method. Practicality of the liposome-based signal amplification method for CRP was also validated by detecting the analyte in the biologically complex matrix. Different concentrations of CRP from 0 ng/mL to 500 ng/mL were spiked in 20% human blood serum and were tested by liposome-based method. As shown in Figure S5, the PGM signal in the real sample was proportional to the concentration of CRP, and the detection limit was calculated to be 13 ng/mL (0.11 nM), which completely meets the clinical demand. These results clearly show the practicality of the proposed liposome-based method for detection of proteins in real biological samples. These results clearly demonstrate that the liposome-based signal amplification method can allow sensitive, selective, accurate, and portable quantitation of different proteins with PGM using different recognition ligands, including both antibodies and aptamers.

Conclusions In conclusion, an enzyme-encapsulated liposome-linked immunosorbent method was developed using personal glucose meters for sensitive, selective, portable and quantitative


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

detection of disease-associated protein biomarkers. This assay makes use of the advantages of liposomes, which are stable, easy to prepare, and suitable for signal amplification and transduction. By trapping a great amount of glucose- producing enzymes in the liposomes, this method avoids the need for multi-step chemical conjugation, thus saving preparation time. Without sophisticated instruments, this method achieves rapid and portable quantitative detection of disease-associated proteins, thereby enabling medical diagnostics at home or in-field. With this approach, as low as 4.5 nM thrombin was detected with high selectivity; the detection limit can be improved to 1.8 nM by simply prolonging the reaction time of amyloglucosidase with amylose. The result of the thrombin detection in human blood serum established the accuracy and reliability of our method. After replacing the amyloglucosidase with invertase, the detection limit was 1.7 nM, which indicated the versatility of liposome-based signal readout strategy. The liposome-based method was verified to have excellent versatility combined with antibodies in a sandwich structure. The detection of C-reactive protein was carried out and the detection limit was 35 ng/mL, which meets the demand of clinical analysis. The liposome-based method showed good accuracy compared to the traditional ELISA method. The method can potentially be further extended to portable quantitative detection of a variety of proteins by replacing the antibody pairs according to targets. Compared to clinically used ELISA methods, this assay showed potential improvements of its easy-operation and portability, it was of great convenience in the resources-limited area. Considering the wide and established usage of antibodies, as well as the low-cost and simplicity of the PGMs, the method can be applied as an available platform for the sensitive, selective, portable and quantitative detection of diseaseassociate proteins.


20

Page 21 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org: synthesis of lipid phosphoramidite, preparation of liposomes, preparation of functionalized MBs, the validation of the influence of surfactant on enzyme activity, optimization of the portable detection conditions, additional figures.

ACKNOWLEDGMENT We thank the National Basic Research Program of China (2013CB933703), the National Science Foundation of China (21325522, 21422506, 21205100, 21435004,

21325522,

21275122,

21521004),

National

Instrumentation

Program

(2011YQ03012412), and National Found for Fostering Talents of Basic Science (NFFTBS, J1310024) for their financial support.

REFERENCES (1)

Warsinke, A., Point-of-Care Testing of Proteins. Anal. Bioanal. Chem 2009, 393, 1393-

1405. (2)

Price, C. P., Regular Review: Point of Care Testing. Br. Med. J. 2001, 322, 1285-1288.

(3)

Yager, P.; Domingo, G. J.; Gerdes, J., Point-of-Care Diagnostics for Global Health.

Annu. Rev. Biomed. Eng. 2008, 10, 107-144. (4)

Tüdős, A. J.; Besselink, G. A.; Schasfoort, R. B., Trends in Miniaturized Total Analysis

Systems for Point-of-Care Testing in Clinical Chemistry. Lab Chip 2001, 1, 83-95.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(5)

Page 22 of 27

Xiang, Y.; Lu, Y., Portable and Quantitative Detection of Protein Biomarkers and Small

Molecular Toxins Using Antibodies and Ubiquitous Personal Glucose Meters. Anal. Chem. 2012, 84, 4174-4178. (6)

Xiang, Y.; Lu, Y., Using Personal Glucose Meters and Functional DNA Sensors to

Quantify a Variety of Analytical Targets. Nat. Chem. 2011, 3, 697-703. (7)

Xiang, Y.; Lu, Y., An Invasive DNA Approach toward a General Method for Portable

Quantification of Metal Ions Using a Personal Glucose Meter. Chem. Commun. 2013, 49, 585587. (8)

Gu, C.; Lan, T.; Shi, H.; Lu, Y., Portable Detection of Melamine in Milk Using a

Personal Glucose Meter Based on an in Vitro Selected Structure-Switching Aptamer. Anal. Chem. 2015, 87, 7676-7682. (9)

Xiang, Y.; Lan, T.; Lu, Y., Using the Widely Available Blood Glucose Meter to Monitor

Insulin and Hba1c. J. Diabetes Sci. Technol. 2014, 8, 855-858. (10) Zhang, J.; Xiang, Y.; Wang, M.; Basu, A.; Lu, Y., Dose‐ Dependent Response of Personal Glucose Meters to Nicotinamide Coenzymes: Applications to Point ‐ of ‐ Care Diagnostics of Many Non‐Glucose Targets in a Single Step. Angew. Chem. Int. Ed. 2016, 55, 732-736. (11) Xue-tao, X.; Kai-yi, L.; Jia-ying, Z., Highly Sensitive and Portable Mercury (Ii) Ion Sensor Using Personal Glucose Meter. Anal. Methods 2015, 7, 81-85.


22

Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Joo, J.; Kwon, D.; Shin, H. H.; Park, K.-H.; Cha, H. J.; Jeon, S., A Facile and Sensitive Method for Detecting Pathogenic Bacteria Using Personal Glucose Meters. Sens. Actuators, B 2013, 188, 1250-1254. (13) Wang, Q.; Wang, H.; Yang, X.; Wang, K.; Liu, F.; Zhao, Q.; Liu, P.; Liu, R., Multiplex Detection of Nucleic Acids Using a Low Cost Microfluidic Chip and a Personal Glucose Meter at the Point-of-Care. Chem. Commun. 2014, 50, 3824-3826. (14) Wang, Q.; Liu, F.; Yang, X.; Wang, K.; Wang, H.; Deng, X., Sensitive Point-of-Care Monitoring of Cardiac Biomarker Myoglobin Using Aptamer and Ubiquitous Personal Glucose Meter. Biosens. Bioelectron. 2015, 64, 161-164. (15) Zhang, J.; Xiang, Y.; Novak, D. E.; Hoganson, G. E.; Zhu, J.; Lu, Y., Using a Personal Glucose Meter and Alkaline Phosphatase for Point‐of‐Care Quantification of Galactose‐1‐ Phosphate Uridyltransferase in Clinical Galactosemia Diagnosis. Chem. - Asian J. 2015, 10, 2221-2227. (16) Wang, Q.; Wang, H.; Yang, X.; Wang, K.; Liu, R.; Li, Q.; Ou, J., A Sensitive One-Step Method for Quantitative Detection of Α-Amylase in Serum and Urine Using a Personal Glucose Meter. Analyst 2015, 140, 1161-1165. (17) Yan, L.; Zhu, Z.; Zou, Y.; Huang, Y.; Liu, D.; Jia, S.; Xu, D.; Wu, M.; Zhou, Y.; Zhou, S., Target-Responsive “Sweet” Hydrogel with Glucometer Readout for Portable and Quantitative Detection of Non-Glucose Targets. J. Am. Chem. Soc. 2013, 135, 3748-3751. (18) Xiong, X.; Wu, C.; Zhou, C.; Zhu, G.; Chen, Z.; Tan, W., Responsive DNA‐Based Hydrogels and Their Applications. Macromol. Rapid Commun. 2013, 34, 1271-1283.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

(19) Torchilin, V. P., Recent Advances with Liposomes as Pharmaceutical Carriers. Nature Reviews Drug Discovery 2005, 4, 145-160. (20) Edwards, K. A.; Baeumner, A. J., Liposomes in Analyses. Talanta. 2006, 68, 1421-1431. (21) Jesorka, A.; Orwar, O., Liposomes: Technologies and Analytical Applications. Annu. Rev. Anal. Chem. 2008, 1, 801-832. (22) Ruktanonchai, U.; Nuchuchua, O.; Charlermroj, R.; Pattarakankul, T.; Karoonuthaisiri, N., Signal Amplification of Microarray-Based Immunoassay by Optimization of Nanoliposome Formulations. Anal. Biochem. 2012, 429, 142-147. (23) Liu, Q.; Boyd, B. J., Liposomes in Biosensors. Analyst 2013, 138, 391-409. (24) Bui, M.-P. N.; Ahmed, S.; Abbas, A., Single-Digit Pathogen and Attomolar Detection with the Naked Eye Using Liposome-Amplified Plasmonic Immunoassay. Nano Lett. 2015, 15, 6239-6246. (25) Zhao, Y.; Du, D.; Lin, Y., Glucose Encapsulating Liposome for Signal Amplification for Quantitative Detection of Biomarkers with Glucometer Readout. Biosens. Bioelectron. 2015, 72, 348-354. (26) Qu, B.; Guo, L.; Chu, X.; Wu, D.-H.; Shen, G.-L.; Yu, R.-Q., An Electrochemical Immunosensor Based on Enzyme-Encapsulated Liposomes and Biocatalytic Metal Deposition. Anal. Chim. Acta 2010, 663, 147-152. (27) Keefe, A. D.; Pai, S.; Ellington, A., Aptamers as Therapeutics. Nature Reviews Drug Discovery 2010, 9, 537-550.


24

Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28) Sun, H.; Tan, W.; Zu, Y., Aptamers: Versatile Molecular Recognition Probes for Cancer Detection. Analyst 2016, 141, 403-415. (29) Wang, X.; Song, P.; Peng, L.; Tong, A.; Xiang, Y., Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging. ACS applied materials & interfaces 2016, 8, 609-616. (30) Singh, A. K.; Kilpatrick, P. K.; Carbonell, R. G., Application of Antibody and Fluorophore ‐ Derivatized Liposomes to Heterogeneous Immunoassays for D ‐ Dimer. Biotechnol. Prog. 1996, 12, 272-280. (31) Wang, Y.; Liu, B., Conjugated Polyelectrolyte-Sensitized Fluorescent Detection of Thrombin in Blood Serum Using Aptamer-Immobilized Silica Nanoparticles as the Platform. Langmuir 2009, 25, 12787-12793. (32) Striggow, F.; Riek, M.; Breder, J.; Henrich-Noack, P.; Reymann, K. G.; Reiser, G., The Protease Thrombin Is an Endogenous Mediator of Hippocampal Neuroprotection against Ischemia at Low Concentrations but Causes Degeneration at High Concentrations. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 2264-2269. (33) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W., Label‐Free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer‐Based Sensor. Angew. Chem. 2005, 117, 5592-5595. (34) Zhao, Q.; Li, X.-F.; Shao, Y.; Le, X. C., Aptamer-Based Affinity Chromatographic Assays for Thrombin. Anal. Chem. 2008, 80, 7586-7593.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

(35) Wang, W.; Chen, C.; Qian, M. X.; Zhao, X. S., Aptamer Biosensor for Protein Detection Based on Guanine-Quenching. Sens. Actuators, B 2008, 129, 211-217. (36) Epstein, F. H.; Gabay, C.; Kushner, I., Acute-Phase Proteins and Other Systemic Responses to Inflammation. N. Engl. J. Med. 1999, 340, 448-454. (37) Ridker, P. M., Clinical Application of C-Reactive Protein for Cardiovascular Disease Detection and Prevention. Circulation 2003, 107, 363-369.


26

Page 27 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only


27

Enzyme-Encapsulated Liposome-Linked Immunosorbent Assay

Recommend Documents