Efficient Hydrogen-Generation CuO/Co3O4 Heterojunction Nanofibers for Sensitive Detection of Cancer Cells by Portable Pressure Meter

Article abstract of DOI:10.1021/acs.analchem.7b01951

Portable, low-cost, and quantitative detection of cancer cells at home and in the field has the potential to revolutionize medical diagnostics. We first report the design and synthesis of highly efficient folic-acid-conjugated hydrogen-generation tube-in-tube CuO/Co₃O₄ heterojunction nanofibers for highly sensitive and rapid recognition of cancer cells through a pressure signal under visible-light irradiation. The resultant nanofibers can dramatically enhance the hydrogen-generation activity of ammonia borane under visible-light irradiation. Such hydrogen-generation reaction can translate a molecular recognition event between folic acid and folate receptor to measurable pressure signal readout through a low-cost and portable pressure meter for target cancer cell detection. Limits of detection (LODs) down to 50 cells mL^-1 in only 15 min can be achieved. This result is superior to those of the other reported methods, indicating the superiority of the new pressure-based sensor in terms of sensitivity. The present study establishes the pressure meter as a useful tool for early clinical point-of-care cancer diagnosis.

Full text of DOI:10.1021/acs.analchem.7b01951

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Article
An Efficient Hydrogen-Generation CuO/Co3O4 Heterojunction Nanofibers
for Sensitive Detection of Cancer Cells by Portable Pressure Meter
Erli Ding, Jun Hai, Tianrong Li, Jie Wu, Fengjuan Chen, Yin Wen, Baodui Wang, and Xiaoquan Lu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01951 • Publication Date (Web): 06 Jul 2017
Downloaded from http://pubs.acs.org on July 6, 2017
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An Efficient Hydrogen-Generation CuO/Co3O4
Heterojunction Nanofibers for Sensitive
Detection of Cancer Cells by Portable Pressure
Meter
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Erli Ding†, Jun Hai†, Tianrong Li†, Jie Wu†, Fengjuan Chen†, Yin Wen§, Baodui Wang†,*, and
Xiaoquan Lu‡,*
†Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province
and State Key Laboratory of Applied Organic Chemistry Lanzhou University Gansu, Lanzhou,
730000 (P.R. China) Lanzhou University, Lanzhou 730000, P. R. China.
‡Department of Chemistry, Tianjin University, Tianjin 300072, P. R. China.
§Department of Pharmacy, Lanzhou University Second Hospital, Lanzhou, Gansu, Lanzhou
730000, P. R. China.
ABSTRACT
Portable, lowꢀcost, and quantitative detection of cancer cells at home and in the field has the
potential to revolutionize medical diagnostics. We first report the design and synthesis of highly
efficient folic acidꢀconjugated hydrogenꢀgeneration tubeꢀinꢀtube CuO/Co3O4 heterojunction
nanofibers for highly sensitive and rapidly recognition of cancer cells through pressure signal
under visible light irradiation. The resultant nanofibers can dramatically enhance the hydrogenꢀ
generation activity of ammonia borane under visible light irradiation. Such hydrogenꢀgeneration
reaction can translate a molecular recognition event between folic acid and folate receptor to
measurable pressure signal readout through a lowꢀcost and portable pressure meter for target
cancer cell detection. Limit of detections (LODs) down to 50 cells mLꢀ1 in only 15 mins can be
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achieved. This result is superior to those of the other reported methods, indicating the superiority
of the new pressureꢀbased sensor in terms of sensitivity. The present study establishes the
pressure meter as a useful tool for early clinical pointꢀofꢀcare cancer diagnosis.
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Graphic
The development of portable sensors for rapid, onꢀsite, and costꢀeffective detection of cancer
cells is of vital significance to early diagnosis and timely prognosis of cancer.1,2 Recently,
optical,3ꢀ7 electrochemical,8ꢀ12 magnetic,13,14 and mass spectrometric15,16 assays for signal
readout have been developed to detect the expression of cell surface receptors on cancer cells
and generally regarded as the gold standards in laboratory scenarios. However, these assays have
high cost, are affected by environmental interferences, and require bulk instrumentation and
services of professional operators. Thus, their use in field work or specific pointꢀofꢀcare testing is
restricted. Therefore, the development of portable sensors for rapid, onꢀsite, and costꢀeffective
detection of cancer cells is necessary.
In recent decades, alternative approaches based on totally different signaling principles using
thermometer, barometer, and hygrometer readouts have been developed for simple and portable
pointꢀofꢀcare testing.17,18 For example, pressureꢀbased bioassays, as signaling techniques, have
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been successfully used to detect protein biomarker.17 In such bioassays, the key step to highly
sensitive detection is the conversion of a biomolecular recognition event into a highlyꢀefficient
gasꢀgeneration reaction. In addition, the reactant and product are nontoxic and environmentally
friendly. Among all existing and emerging gasꢀgeneration reactions, ammonia borane (NH3BH3,
denoted as AB) decomposition has attracted extensive concern because of the high hydrogen
capacity of AB (19.6 wt.%). Notably, this process generates nontoxic H2. Theoretically, the
decomposition of 1 mmol of AB generates 67.2 mL of H2 under standard conditions. If this
solution is confined to a 1ꢀmL tube, the pressure increases to over 6×106 Pa, which can be
detected easily by a pressure meter. However, such a promising gasꢀgeneration reaction is rarely
used for pressureꢀbased bioassays. To date, many noble metals, such as Pd and Au,19 exhibit
highly efficient catalytic activity for AB dehydrogenation. However, their high cost, poor
durability, and scarcity greatly restrict their use in largeꢀscale commercial applications.
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Semiconductor heterojunction photocatalysts have attracted significant attention20 because
they can achieve optimal solar light absorption and enhance surface reactivity. The catalytic
activities of heterojunction nanomaterials are often superior to those of constituent elements
because of their efficient exploitation of photoꢀgenerated electron/hole (e/h+) pairs. This process
accounts for the enhanced performance of heterojunction nanomaterials in relevant
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photoconversion processes. Among various oxide semiconducting materials, CuO21 and Co3O4
photocatalysts have been the most wellꢀknown. Such oxideꢀbased photocatalysts have been
extensively studied as replacement for noble metalꢀbased catalysts. We hypothesized that using
such photocatalyst could significantly accelerate the reaction rate of AB dehydrogenation. This
gasꢀgeneration photocatalyst can be linked with a molecular recognition event as a molecular
recognition tool to construct a simple, rapid, sensitive, affordable, and specific cancer cell
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detection method.
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Scheme 1. Synthetic route of folic acid (FA)ꢀconjugated tubeꢀinꢀtube CuO/Co3O4 heterojunction nanofibers
and its working principle for detection of cancer cells. The strong driving force elicited by specific interactions
between overexpress folate receptor (FR) and FA caused the conjugation of functionalized CuO/Co3O4NFs
onto the surface of the cancer cells. When added the ammonia borane ( AB), hydrogen (H2) can generate in the
bottle, leading to a significant pressure increase in the reaction chamber allowing a sensitive readout by using a
portable pressure meter.
In this study, we proposed a twoꢀstep and facile route to fabricate tubeꢀinꢀtube CuO/Co3O4
heterojunction nanofibers (NFs) and demonstrated its usage in cancer detection based on
pressure signal. The resulting heterojunction NFs exhibits excellent catalytic activity during AB
hydrolysis for hydrogen generation. On the basis of these findings, we use catalyzed gasꢀ
generation reaction as signal readout for highly sensitive detection of cancer cells. The approach
is based on the folateꢀconjugated CuO/Co3O4NFs, which provide dual functionality by binding to
folate receptors, expressing cancer cells, and facilitating detection through the photocatalytic
decomposition of the high hydrogen capacity of AB. This dual functionality results in significant
pressure increase in the reaction chamber. The increase can be sensitively detected by a simple,
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low cost, and portable pressure meter (scheme 1). To our best knowledge, this study is the first to
use a pressure meter that detects the target cells simply and portably on the basis of AB
decomposition.
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EXPERIMENTAL SECTION
Materials. Polyacrylonitrile (PAN), polyvinylpyrrolidone (PVP), cupric acetate and cobalt (II)
aetate tetrahydrate were all purchased from Macklin. Ammonia borane was purchased from
HWRK CHEM. All the chemical agents were used as received without further purification.
PEGꢀ3,4ꢀdihydroxy benzyl amine (DIBꢀPEGꢀNH2) and PEGꢀ3,4ꢀdihydroxy benzyl amine ꢀfolic
acid (DIBꢀPEGꢀNHꢀFA) were synthesised according to our previous work. [31]
Measurements. The morphology of the samples was investigated by fieldꢀemission scanning
electron microscope (FEꢀSEM, FEI, Sirion 200). TEM measurements were carried out with a
JEMꢀ2100 (200 kV) instrument under ambient conditions through the deposition of ethanol
dispersions of the nanomaterials on amorphous carbonꢀcoated copper grids. Xꢀray powder
diffraction patterns of the particles were recorded using a Bruker AXS D8 Advance
diffractometer with CuKα radiation (l=1.5418 Å). Xꢀray photoelectron spectroscopy (XPS)
measurements were performed using a PHIꢀ5702 multifunctional spectrometer with AlKα
radiation. UVꢀVisꢀNIR absorption spectra were carried out on a Agilent Cary 5000 UVꢀVisꢀNIR
Spectrophotometer. Photocatalysis was performed using Xenon lamp (HSXꢀF/UV 300),
equipped with 400ꢀ800 nm filter. FTꢀIR characterization was carried out on a Bruker Vertex 70
FTꢀIR
spectrometer.
Fluorescence microscopic images were viewed under a fluorescent microscope (Leica DR) equip
ped with a digital camera (ORCAꢀER, Hamamatsu). Cyclic voltammetry (CV) measurements
were obtained by using CHI 760E electrochemical workstation. Glassy carbon and Pt electrodes
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were used as working and counter electrodes, respectively. Ag/AgCl electrode worked as
reference electrode. Cu and Co contents are determined by inductively coupled plasmaꢀatomic
emission spectrometry (ICPꢀAES, Varian VISTAꢀMPX).
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The synthesis of CuO/Co3O4 heterojunction nanofibers. The preparation of CuO/Co3O4 1D
nanostructures involves two steps. Briefly, a mixture of 1 g PVP, 1 g PAN, 1.5 mmol
Co(CH3COO)2.4H2O, 1.5 mmol Cu(CH3COO)2·H2O and 10 ml DMF was stirred at room
temperature for 24 h, and then a viscous solution was obtained. Next, the precursor solution was
loaded into a plastic syringe with a stainless steel nozzle (8#), which was connected to a highꢀ
voltage power supply. A high voltage of 18 kV was applied between the needle tip and
aluminum foil collector with a distance of 17 cm and the spinning rate was controlled at 1 ml hꢀ1.
0
Finally, the asꢀspun fibers were calcinated at 400 ℃ for 2 h at a heating rate of 4 C minꢀ1. The
other rate catalyst was synthesized by controlling the rate of salts added.
The synthesis of folic acid-functionalized CuO/Co3O4NFs. 0.02 g DIBꢀPEGꢀNH2 and 0.002 g
DIBꢀPEGꢀNHꢀFA33 was added to 5 mL water solution containing 0.02 g CuO/Co3O4 NFs. After
stirred for 12 h in the dark at room temperature, the mixtures were collected by centrifugation,
washed with water, and then dispersed in water and dialyzed with H2O for 24 h to remove
unreacted organic molecules.
Catalytic Decomposition of Aminodiborane. To study the catalytic properties of the asꢀ
synthesized products, their catalytic activities were evaluated for the dehydrogenation of AB in
aqueous solution under light or dark. In a typical experiment, 0.014 mmol catalyst and 3.3 mL
water was added into a double walled glass tube with the neck connected to a gas burette through
soft rubber tube. After stirring for 5 mins, 0.667 mmol of NH3BH3 was quickly added into the
0
glass tube. The sealed reaction solution was stirred under light at 25 ± 1 C (by using a
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connected reflux water condenser). The volume of hydrogen gas generated from the hydrolysis
reaction was measured using the waterꢀdisplacement method. As a control, the same reaction
was conducted in dark condition at room temperature. Recycling of CuO/Co3O4 NFs catalyst was
tested under light at room temperature in water (3.3 mL) containing 0.028 mmol catalyst, with
addition of AB (0.667 mmol) to the system in each cycle.
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The apparent quantum efficiency (AQE) measurment. The apparent quantum efficiency
(AQE) measurement: A 300ꢀW Xe arc lamp was used as the light source for photocatalytic
reaction. The measurement of AQE was performed using same amount reactions. We fixed the
wavelength at 450 ± 10 nm for Vis irradiation. The laser power in the photocatalytic reaction
was collected using a power meter (Newport; 843ꢀR). The corresponding wavelengths captured
for AQE calculation are located at 450 ± 10 nm. Thus, the AQE was calculated as the following
equation, AQE =n/np × 100 %, in which n and np were denoted as the number of photos that
generating product needed and the number of incident photons, respectively.
The apparent quantum efficiency (AQE) caculation. Under visible light irradiation (450 ± 10
nm) for CuO/Co3O4NFs within 1 min. The average intensity of irradiation was found to be 0.255
W and 0.072 W before and after the catalyst added to reaction flask by a power meter (Newport;
843ꢀR). Total absorb light energy ꢁE =(0.255 – 0.072) × 1 × 60 = 10.98 J, n = 0.0344 mmol,
energy per photon E0 = hc/λ =6.63 × 10ꢀ34 × 3 × 108/(450 × 10ꢀ9) = 4.42 × 10ꢀ19 J, molor of
photons np =ꢁE/(E0 × NA) = 10.98/(4.42 × 10ꢀ19 × 6.02 × 1023) = 0.0413 mmol, AQE (initial) =
n/np = 0.0344/0.0413 × 100 % = 83.3 %. With the same method, we can calculate the initial
AQE for other catalysts, respectively.
Cyclic voltammetry (CV) analysis. To investigate the redox property of CuO, Co3O4 and CuO/
Co3O4, CV analysis was performed with a standard threeꢀelectrode system, which consists of an
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Ag/AgCl reference electrode (0.01 M AgNO3, 0.1 M TBAPF6 in CH3CN), a platinum sheet
working electrode and a platinum wire counter electrode. The electrolyte solution employed was
0.10 M TBAPF6 in freshly dried CH3CN, and ferrocene/ferrocenium redox coupled (Fc/Fc+, ꢀ
4.8 eV) was used as the reference material for all CV measurements. CV was recorded at 100
mV/s under a N2 atmosphere.
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Cytotoxicity assay and bioassay assay. In vitro cytotoxicity of the CuO/Co3O4NFs was
evaluated by performing a methyl thiazolyl tetrazolium (MTT) assay of the HeLa cells incubated
with the particles. Cells were seeded into a 96ꢀwell cell culture plate with a density of 5 × 103
cells per well in DMEM with 10% FBS at 37 °C under 5% CO2 for 24 h. Then, the cells were
incubated with the CuO/Co3O4NFs with different concentrations (0, 23, 34, 45, 56, 62 and 68 ꢂg
mLꢀ1 in DMEM) for 24 h and 48 h at 37 °C under 5% CO2. Thereafter, MTT (10 mL, 5 mg mLꢀ1)
was added to each well and the plate was incubated for 4 h at 37 °C. After the addition of
dimethyl sulfoxide (DMSO, 100 ꢂL per well), the cell plate was allowed to stand at 37 °C for 15
mins. The optical density was measured at 492 nm using a microplate reader (Shanghai Sanco
Instrument Co.,Ltd 318Cꢀmicroplate reader).
HeLa cells, A549 cells, and NIH3T3 cells were maintained at 37 ºC, 5% CO2 in a humidified
incubator. Cells were plated in 6 well plate at density of 1 × 105 cells per well. After 24 h, cells
were treated with different concentration of FAꢀCFO/Co3O4 NFs (23 to 68 ꢂg mLꢀ1) for 1 h.
Afterwards cells were washed with PBS buffer, the cells and 5mg AB were transferred to a 2ml
tube and kept the liquid volume up to 1 mL. The pressure change in the tube was monitored by a
portable homeꢀmade pressure meter under the visible light (LED lamp as the light source) for 15
mins. In another set of experiment, similar immunoassay were carried out using increasing
number of HeLa cells (50 to 105 cells) keeping the concentration of FAꢀCuO/Co3O4 NFs constant
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(23 ꢂg mLꢀ1).
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Detection of cancer cells in whole blood. HeLa cells were first spiked into healthy mouse or
human blood to prepare artificial whole blood samples with concentrations of about 50, 102,
2×102, 3×102, 4×102, 5×102, 103, and 104 cells mLꢀ1. Then, 68 ꢂg mLꢀ1 of FAꢀCFO/Co3O4 NFs
was added into the above blood samples and incubated for 1h. Afterwards blood samples were
washed with PBS buffer, the blood samples and 5mg AB were transferred to a 2 ml tube and
kept the liquid volume up to 1 mL. The pressure change in the tube was monitored by a portable
homeꢀmade pressure meter under the visible light (LED lamp as the light source) for 15 mins. In
order to identify that HeLa cells existed in whole blood sample, the threeꢀcolor
immunocytochemistry method was used. First, HeLa cells and blood cells were sequentially
treated with 4% paraformaldehyde in PBS for 20 mins and 20 µL of methanol for 10 mins. Then,
the cells were sequentially stained with 50 µL of antiꢀCD45 stock solution (50 µL of antibody
stock solution in 1 mL of PBS) for 45 mins, 50 µL of antiꢀCK stock solution (50 µL of antibody
stock solution in 1 mL of PBS) for 45 mins, and 20 µL of Hoechst solution (2 µg mLꢀ1 Hoechst
in PBS) for 15 mins, respectively. Finally, cells were imaged by using Leica DR microscope.
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RESULTS AND DISCUSSION
Synthesis and Characterization of the Catalyst. CuO/Co3O4NFs were synthesized using a
certain amount of Co(CH3COO)2/Cu(CH3COO)2, polyvinylpyrrolidone (PVP), and
polyacrylonitrile (PAN) by a simple method involving two steps, namely, electrospinning and
thermal treatment. As shown in Figure 1, the formed CuO/Co3O4NFs perfectly maintained the
1D nanostructure. In addition, compared with PAN@PVP/Co(CH3COO)2/Cu(CH3COO)2
composite NFs, the formed CuO/Co3O4NFs had a rough surface with many nanoparticles (NPs),
which formed a unique tubular structure. The formed CuO/Co3O4NFs were further confirmed by
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the transmission electron microscope (TEM) images (Figure 1C and D). The CuO/Co3O4NFs
were composed of NPs, and each nanoparticle was attached to several other NPs. Many
nanopores were observed to be within the spaces between the adjacent NPs. Apparently, the dual
tubular structure of the CuO/Co3O4 NFs possessed wellꢀseparated walls along nearly their entire
length, and the average diameter of the outer tubes and inner tube was approximately 210 and
100 nm, respectively (Figure 1C). Meanwhile, the highꢀresolution HRTEM images of the
CuO/Co3O4NFs are shown in Figure 1D. The simultaneous presence of crystalline Co3O4 and
CuO crystal lattices were observed in the CuO/Co3O4NFs. An intimate contact between CuO and
Co3O4 was also observed. Figure 1D shows that an interplanar distance of 0.47 nm can be
indexed as the interplanar distance of the Co3O4 (111) planes.23 The interplanar distances of
0.235 nm agreed well with the lattice spacing of the (111) planes of the hexagonal wurtzite
CuO.24 These results confirmed that heterostructures were formed between Co3O4 and CuO NPs
in the CuO/Co3O4NFs. The local composition of the resulting CuO/Co3O4NFs was also
examined through high angle annular darkfield scanning transmission electron microscopy
(HAADFSTEM), corresponding elemental mapping (Figure 1E–H), and energyꢀdispersive Xꢀray
spectroscopy (EDX; Figure S1). The results clearly showed the presence of Cu, Co, and O
elements.
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Figure 2A shows the XRD patterns of the asꢀsynthesized nanofibers. The CuO/Co3O4 NFs had
two sets of diffraction peaks, which were correspondingly ascribed to the cubic spinel Co3O4
(JCPDS card no. 01ꢀ076ꢀ1802) and CuO phase (JCPDSꢀICDD 05ꢀ0661). No characteristic peaks
for impurity, such as CuCo2O4, were observed, suggesting that Co3O4 and CuO formed
heterojunction in the nanofibers. Moreover, typical Xꢀray photoelectron spectroscopy (XPS)
spectra (Figure S2) showed the presence of Co, Cu, O, and adventitious C in the CuO/Co3O4
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nanofibers. As observed in Figure S2b, two peaks at binding energies of 933.9 and 953.9 eV in
Cu 2p XPS spectra could be assigned to Cu 2p3/2 and Cu 2p1/2, respectively. The peaks shift
0.3 eV compared to CuO. 25 The above values indicate a normal state of Cu2+ in the CuO/Co3O4
nanofibers. As for Co peaks, the binding energies located at ~780.4 and ~795.5 eV can be
assigned to Co 2p3/2 and Co 2p1/2 (Figure S2c), together with a spinꢀenergy separation of
around 15 eV, demonstrating the presence of a Co3O4 phase. 26 Nitrogen adsorption measurement
results (Figure 2B) revealed that the CuO/Co3O4NFs exhibited type IV isotherm behavior with
H3 hysteresis, implying that the obtained fibers were mesoporous. The distribution of Barrettꢀ
JoynerꢀHalenda pore sizes determined from the adsorption branches peaked at ~25 nm.
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Figure 1. SEM images of precursor NFs (A) and CuO/Co3O4 NFs (B); C) TEM image and SAED pattern of
CuO/Co3O4 NFs; D) HRTEM images of CuO/Co3O4 NFs; E) The corresponding STEMꢀEDS elemental
mapping images of Cu (F), Co (G), and O (H) in CuO/Co3O4 nanofibers.
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Figure 2. A) XRD patterns of CuONFs, Co3O4NFs, and CuO/Co3O4NFs; B) N2 adsorption/desorption
isothermals of CuO/Co3O4 NFs; C) UVꢀvis spectra of CuONFs, Co3O4NFs, and CuO/Co3O4NFs; D) Band
diagram and mechanism of charge separation for pꢀCo3O4/pꢀCuO heterojunction.
Figure 2C shows the UVꢀvis spectra of the CuONFs, Co3O4NFs, and CuO/Co3O4NFs. The
absorption edges of the CuONFs appeared at 832 nm and corresponded to the band gap energies
of 1.49 eV. The Co3O4NFs exhibited two main absorption bands, and the absorption edges
appeared at 663 nm and corresponded to the band gap energies of 1.87 eV. These values were
close to the reported values of CuO (1.40 eV) and Co3O4 (1.89 eV).27 The CuO/Co3O4NFs
indicated that two evident absorption bands were present, and these bands were ascribed to the
characteristic absorption of CuO and Co3O4. We estimated the conduction bands, valence band
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edges, and Eg values of CuO and Co3O4 (Figure 2D) according to the cyclic voltammetry (Figure
S4) test and Tauc plots (Figure S3). Since the value of ECB for Co3O4 is more negative than that
of CuO, under visible light illumination, the excited electrons of Co3O4 can be transported to the
conduction band of CuO. While the photogenerated holes on the valence of Co3O4 cannot
transfer to that of CuO, so the combination of electrons and holes can be prevented.28 As a
consequence, the separation of photogenerated electronꢀhole pairs is significantly improved.
Thus, we proposed a mechanism of charge separation for CuO/Co3O4 heterojunction (Figure 2D).
Catalytic Properties and Mechanism. The prepared CuO/Co3O4NFs were first tested for the
catalytic hydrolysis of AB to generate H2 under dark and visible light irradiation (λ > 420 nm) at
room temperature. The activities of the catalysts under light were enhanced to a greater extent
compared with those under dark environment (Figure 3A). Under light, the reaction can be
completed within 4.5 mins, which is faster than the time undertaken by the same reaction under
dark environments (8 mins). The contribution of the thermal effects induced by visibleꢀlight
irradiation was eliminated at the same reaction temperature (25 ± 1 °C) for AB hydrolysis (by
using a connected reflux water condenser) under dark conditions and visibleꢀlight irradiation.
Under visible light irradiation, the photocatalytic property of the CuO/Co3O4NFs toward AB
hydrolysis was tested and compared with those of CuONFs, Co3O4NFs, and physical mixture of
CuO NFs and Co3O4 NFs at 25 ± 1 °C. Figure 3B shows the amount of H2 generated as a
function of reaction time. Evidently, the H2 generation rates of the CuO/Co3O4 catalyst were
enhanced to a greater extent compared with those of their monometallic or mixture counterparts.
Simultaneously, a quantum efficiency of 83.3% at 450 ± 10 nm was achieved by the
CuO/Co3O4NFs. This efficiency was the highest among the efficiencies of all asꢀprepared
nanofibers (Figure S6). The highest photoactivity can be ascribed to the formation of the
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nanojunction between the CuO and Co3O4, and the generated electrons enriched on the
conduction band of Co3O4 enhanced the photocatalytic activity of the CuO/Co3O4 catalyst for the
AB dehydrogenation (Figures S7 and 8). In addition, the resulting CuO/Co3O4 catalyst had high
cycle stability (Figures S9, 10, and 11).
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Figure 3. A) Hydrogen evolution in aqueous solution(3.3 mL) containing fixed amount of AB (0.65 mmol)
and CuO/Co3O4NFs(0.014 mmol) under or without light at 298 K; B) Relationship between dehydrogenation
rate of AB (0.65 mmol) and different catalysts (0.014 mmol) at 298 K in water(3.3 mL) under light.
Cytotoxicity assay and bioassay assay. Given the high photocatalytic activity towards AB
hydrolysis, CuO/Co3O4NFs was designed as a sensing platform for highly sensitive detection of
cancer, which was based on pressure response. In order to improve the dispersity of
CuO/Co3O4NFs in water and enhance the targeting ability of the CuO/Co3O4NFs for cancer cells,
we used the PEGꢀ3, 4ꢀdihydroxy benzyl amine (DIBꢀPEGꢀNH2) and PEGꢀ3, 4ꢀdihydroxy benzyl
amineꢀfolic acid (DIBꢀPEGꢀNHꢀFA) to modify the CuO/Co3O4NFs (Scheme 1). The detailed
characteristics were provided in the supporting information (Figures S12 and 13). Folic acid is a
highꢀaffinity ligand for the folate receptor (FR), and has been widely used for targeted delivery
of FAꢀconjugated molecular probes or nanoparticles to FRꢀoverexpressing cancer cell lines (e.g.,
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HeLa and KB cell lines).29,30 Cytotoxic activity was evaluated before the application of
CuO/Co3O4NFs for cancer cell detection. As shown in Figure S14, the cell viability of
CuO/Co3O4NFs for HeLa cells remained more than 75% upon incubation with CuO/Co3O4NFs
at 68 ꢂg mLꢀ1 concentration for 48 h, indicating the low cytotoxicity of the CuO/Co3O4NFs.
HeLa cells and A549 cells that overexpress folate receptor were used to test the performance of
the selectivity of FAꢀCuO/Co3O4NFs.29 In the control experiments, we used NIH3T3 cells that
do not express folate receptors.31 Three kinds of cells were incubated with FAꢀCuO/Co3O4NFs
for 1 h. The cells were then washed with 1× PBS, and incubated with AB (5 mg). The pressure
change in the tube was monitored under visible light by a portable homeꢀmade pressure meter.
As shown in Figure 4A, HeLa and A549 cells with the same cell number (6×105) exhibited
significant pressure change, whereas the NIH3T3 cells provided only negligible signals. This
result clearly indicated that the discrimination between FAꢀpositive cancerous cells and normal
cells can be easily recognized through pressure changes.
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Figure 4. Pressure determination of the expression of the folate receptor in cancer cells using
FAꢀCuO/Co3O4NFs. A) Pressureꢀchange profiles of cells after incubation with FAꢀ
CuO/Co3O4NFs for 1 h and then addition of AB. B) quantitatively detect HeLa cells using FAꢀ
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CuO/Co3O4 NFs and AB based on pressureꢀchange. Values are expressed as mean ±SD.
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We then investigated the limit of detection (LOD) and detection dynamics of the biosensor
assay. HeLa cells at varying concentrations were incubated with FAꢀCuO/Co3O4 NFs. As shown
in Figure 4B, with the increase of cells number, the measured ꢁP continuously increased.
Moreover, the ꢁP against cells number showed a good linear correlation, realizing an LOD of 50
cells mLꢀ1. The LOD obtained was lower than those obtained by other reports,32 which
demonstrating the excellent sensitivity of FAꢀCuO/Co3O4NFs for quantitative bioanalysis. The
detection dynamic curve displayed in Figure S15 indicated that the gas pressure increases over
incubation time. However, the incubation time of 15 mins was basically sufficient to
discriminate the interactions between the FAꢀCuO/Co3O4NFs and HeLa cells. Therefore, such
pressure change detection platform is well suited for highly sensitive and rapid cancer detection
in clinical and pointꢀofꢀcare settings.
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Figure 5. Confocal images of HeLa cells alone (A), HeLa cells incubated with CuO/Co3O4 for 1 h
(B), NIH3T3 cells alone (C), and NIH3T3 cells incubated with CuO/Co3O4 for 1 h (D).
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We also analyzed the deposition of CuO/Co3O4NFs on the cell surface. Confocal images were
used to determine whether CuO/Co3O4NFs are present on the cell surface. Figure 5A, 5B, and
S16 shows that the strong driving force elicited by specific interactions between overexpress
folate receptor and FA caused the conjugation of functionalized CuO/Co3O4NFs onto the surface
of the HeLa cells in the presence of the target cells. Moreover, no nanofiber precipitate was
detected on the surface of the negative control cells (Figure 5 C and D).
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Figure 6. Detection performance of FAꢀCuO/Co3O4NFs for artificial whole blood samples spiked with HeLa
cells. The capture efficiency of prestained HeLa cells is achieved on antiꢀEpCAMꢀcoated HTiNS coatings for
artificial whole blood samples with concentration of 50, 102, 2×102, 3×102, 4×102, 5×102, 103, and 104 cells
mLꢀ1. Values are expressed as mean ±SD.
To demonstrate the potential clinical application, the sensor was also used to detect HeLa in
artificial whole blood. Briefly, we prepared the artificial whole blood samples by spiking healthy
mouse blood with HeLa cells at concentrations of approximately 50, 102, 2×102, 3×102, 4×102,
5×102, 103, and 104 cells mLꢀ1. As shown in Figure 6, the measured ΔP also increased linearly
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with the cells number, an LOD of 50 cells mLꢀ1 was obtained. Such a sensitive detection method
was confirmed in human blood samples by spiking healthy human blood with HeLa cells (Figure
S16). When the CuO/Co3O4 NFs kept in blood and stayed for 24 hours, we checked the stability
of CuO/Co3O4 NFs by using the XRD and SEM. As shown in Figure S17, there is not change of
composition and morphology. Therefore, the reagent is stability in detection system. In addition,
we employed the threeꢀcolor immunocytochemistry method based on PEꢀlabeled antiꢀCK (CK, a
protein marker for epithelial cells), FITCꢀlabeled antiꢀCD45 (CD45, a marker for white blood
cells), and Hoechst for nuclear staining to confirm the HeLa cells in a artificial whole blood
sample. As shown in Figure 7, HeLa cells can be clearly identified from whole blood sample.
Therefore, these results show the potential biomedical application of hydrogenꢀgeneration
CuO/Co3O4 heterojunction nanofibers based on pressureꢀbased bioassays for early diagnosis and
monitoring of cancer patients.
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Figure 7. Threeꢀcolor immunocytochemistry method for identifying HeLa cells from white blood cells (WBCs)
including Alexa Fluor488ꢀlabeled antiꢀCK (CK, a protein marker for epithelial cells), FITCꢀlabeled antiꢀCD45
(CD45, a marker for WBCs) and Hoechst for nuclear staining. (Scale bar = 20 µm).
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CONCLUSION
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In summary, for the first time, we have designed a simple and general method based on gasꢀ
generation CuO/Co3O4 heterojunction nanofibers and a portable pressure meter for inexpensive,
rapid, portable, userꢀfriendly, and quantitative detection of specific cancer cells. This method
provided an excellent dynamic range of 50 to 105 cells mLꢀ1 for Hela cells with a detection limit
of at least 50 cells mLꢀ1 in only 15 mins. In addition, such pressureꢀbased bioassays can
efficiently detect Hela cells from artificial whole blood samples with high sensitivity. The
excellent performance of the method is attributed to the highly efficient photocatalytic activity of
CuO/Co3O4 NFs during AB dehydrogenation and high hydrogen capacity of AB. To the best of
our knowledge, this is the most sensitive detection method for cancer cells. The approach
proposed in this study has the potential to perform early clinical pointꢀofꢀcare cancer diagnosis.
It can be used in the field and by the public at home because of its simple design and operation,
low cost, portability, and excellent sensitivity and its ability to recognize different cancer cells.
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ASSOCIATED CONTENT
Supporting Information
Characterizations, Supporting Figures and Tables are available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: wangbd@lzu.edu.cn; luxq@tju.edu.cn.
ACKNOWLEDGMENTS
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The work was supported by the National Natural Science Foundation of China (21671088,
21401091, 21431002 and 21501080, 21575115 and 21327005), and Fundamental Research
Funds for the Central Universities (lzujbkyꢀ2017ꢀ105).
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ACS Paragon Plus Environment