共查询到20条相似文献，搜索用时 46 毫秒
1.
The microbicidal capacity of polymorphonuclear leucocytes of diabetic and control subjects was evaluated by estimating the release of lysosomal enzymes viz beta-glucuronidase, lysozyme, acid phosphatase, alkaline phosphatase, in response to a particulate stimulus-serum treated zymosan (STZ). The cells untreated and pretreated with cytochalasin B were exposed to STZ The total enzyme activities were estimated after cell lysis. The total enzyme activities were not altered in diabetic subjects as compared to control subjects. The release of lysosomal enzymes by cells pretreated with cytochalasin B was high as compared to untreated cells. The release of lysosomal enzymes from the cells isolated from diabetic patients, untreated and pretreated with cytochalasin B was reduced as compared to controls. The findings of the present study that the total lysosomal enzyme activities are normal while the release of these enzymes in response to stimulus is impaired in diabetics, suggest that the bactericidal capacity of these cells which involves phagocytosis is impaired in these patients. 相似文献
2.
K. Srikrishna A. S. Kanagasabapathy Lily John 《Indian journal of clinical biochemistry : IJCB》1994,9(1):5-8
The urinary excretion patterns of N-acetyl-β-D-glucosaminidase (NAG), alanine aminopeptidase (AAP) and protein/creatinine ratio (UP/UCR) were studied in 133 diabetic subjects under treatment, 7 patients with established diabetic nephropathy (DN) and 79 carefully selected (age-matched) healthy subjects. NAG, AAP and UP/UCR were highly elevated in DN, while in diabetics urinary NAG levels correlated well with the degree of long-term metabolic control indicated by glycosylated hemoglobin (GHB or Hba1). Both AAP and UP/UCR were found to be more sensitive than NAG, but less specific. Urinary NAG and AAP assays thus offer simple, sensitive and non-invasive techniques for prognostic indication of the onset of microangiopathic changes in long-term diabetic subjects. 相似文献
3.
Harleen Mangat M. P. J. S. Anandaraj A. Anjaneyuiu 《Indian journal of clinical biochemistry : IJCB》1994,9(1):26-30
Calcium—activated neutral proteases (CANP) were examined in the subcellular fractions of the skeletal muscle from Duchenne muscular dystrophy patients and healthy individuals. Both and m CANP were detected in subcellular fractions of skeletal muscle. An increase in the quantity of CANP in subcellular particles of DMD muscle was observed. A 33 fold rise in the concentration of calcium in nuclei of dystrophic muscle was noted followed by cytosol, myofibrils, microsomes and mitochondria. 相似文献
4.
A potent hypoglycaemic principle was isolated by us earlier from the seeds of fenugreek. We have now investigated its hypocholesterolemic effect. Hypercholesterolemia was induced in two groups of rabbits (5 each) by feeding orally cholesterol 100 mg/kg/day for one week. From 8th day group I animals (controls) received the same dose of cholesterol for 4 more weeks. Group II animals (treated) were given along with the same dose of cholesterol fenugreek principle at 50 mg/kg/day for 4 weeks. Fenugreek principle not only prevented the elevation of serum cholesterol, (LDL+VLDL)c, triacylglycerols and the ratios of total cholesterol/HDLc and (LDL+VLDL)c/HDLc, but also brought down most of these values. Blood glucose levels were normal. The study shows that the same hypoglycaemic principle has hypocholesterolemic effect also. 相似文献
5.
M. Jain Kavita Sharma V. P. Sharma 《Indian journal of clinical biochemistry : IJCB》1994,9(2):106-108
Zinc (Zn), copper (Cu), magnesium (Mg) and retinol levels were studied in serum and tissue of the patients with prostatic carcinoma (Ca), benign hyperplasia (BHP) and control subjects. Zinc and retinol levels were low in both serum and tissues of patients with carcinoma, while in BHP, both zinc and retinol level were decreased. Copper levels were high in serum and tissues of both BHP as well as carcinoma patients. Magnesium levels were elevated in both serum and tissues of cancerous patients. The results indicate that Zn, Cu, and Mg metabolism is disturbed in malignancy. 相似文献
6.
The effect of sodium pentosan polysulphate (SPP), was studied in relation to certain blood and erythrocyte membrane parameters in calcium oxalate stone forming rats. Calcium oxalate stones were induced by feeding the rats with 3% w/w sodium glycollate. Fibrinogen, haemoglobin and serum protein levels did not show any variation with the treatment procedures. Serum mucoprotein and protein bound carbohydrates-hexosamine and sialic acid-were increased significantly in the rats receiving calculogenic (CPD) and attained nearly normal levels with SPP treatment. In contrast, hexuronic acid level was decreased in the CPD group and SPP administration increased the level of hexuronic acid in the treated groups. Erythrocyte membrane Ca2+-ATPase activity was increased in stone forming rats and SPP administration brought a reduction in the above enzyme activity. Changes in Membrane Mg2+- and Na+, K+-ATPases were minimal. Membrane cholesterol and phospholipids were also raised significantly in stone formers, SPP treatment reduced the membrane cholesterol levels in both controls and stone formers. Phospholipids were also decreased moderately. The above observations suggest that SPP is safe for administration in urolithiatic condition without adverse effects. 相似文献
7.
G. Kashinath Mira Pal K. Ramachandran P. Bhat 《Indian journal of clinical biochemistry : IJCB》1994,9(2):87-90
Serum phenytoin levels were measured in grand mal epilepsy patients receiving diphenyl hydantoin. The drug levels were correlated with various biochemical parametres. A linear relationship between the levels of diphenyl hydantoin and creatinine was observed. This positive correlation coefficient was found to be statistically signifficant. This correlation may be related to a positive Jaffe’s reaction seen with the chromogen diphenyl hydantoin. 相似文献
8.
J. Baxi Kavita Sharma Rajbala Asha Mehta A. K. Pendse P. P. Singh 《Indian journal of clinical biochemistry : IJCB》1994,9(1):47-49
Evaluation of serum SOD and MDA level was done in 21 first episode renal stone formers, 9 recurrent stone formers, 20 patients with obstructive uropathy other than urolithiasis and 12 patients with urinary infection. Twenty-two healthy volunteers were taken as controls. The level of SOD in respective groups was 2.12±0.84, 2.78±0.85, 1.42±0.31, 1.98±0.70 and 2.32±0.62 units/ml and of MDA was 2.61±1.07, 2.69±1.15, 1.65±0.33, 1.33±0.34 and 1.55±0.48 n mol/ml respectively. The results indicate increased peroxidative stressin nephrolithiasis only. Since SOD level was normal in all groups, this increased peroxidative stress in nephrolithiasis should be due to factors other than this one. 相似文献
9.
D. C. Sharma Raj Kiran Vijaywantee Ramnath Krishna Khushiani P. P. Singh 《Indian journal of clinical biochemistry : IJCB》1994,9(2):100-102
Blood haemoglobin, serum iron, iron binding capacity, transferrin saturation and ferritin levels were determined in two groups of mothers as well as their cords—strict vegetarians (lactovegetarians) and non-vegetarians (omnivores), closely comparable in age, weight, parity and gestation period but differing in their diet and food habits. All these parameters, except total iron binding capacity, were found to be significantly lower in vegetarian mothers and their cords as compared to nonvegetarian mothers and their cords, respectively, despite receiving supplemental iron for six months. Further, there was a greater incidence of anemia and iron deficiency in mothers consuming only vegetarian diet. Moreover, a significant correlation existed between mother's ferritin to cord ferritin confirming that maternal iron deficiency does affect neonatal iron status. All these observations suggest that strict vegetarian mothers as well as their newborns have a greater incidence and risk of anemia and iron deficiency. 相似文献
10.
Farzana Bano A. M. Kidwai R. K. Upreti R. K. Singh 《Indian journal of clinical biochemistry : IJCB》1994,9(2):109-111
The present studies with aluminium chloride given in drinking water showed marked inhibition in the activities of intestinal brush border membrane marker enzymes, namely alkaline phosphatase, acetyl cholinesterase, γ-glutamyl transpeptidase and sucrase. Moreover, a significant depression in the levels of membrane constituents, viz phospholipids, cholesterol, hexoses and sialic acid following aluminium chloride treatment was also observed. On the basis of these studies it may be concluded that if aluminium is taken in high quantities it may alter the structure and functioning of the intestinal brush border membrane, which in turn may lead to the improper digestion and reduced nutrient uptake from the small intestine. 相似文献
11.
S. Biswas A. Bhelwa A. Itoop S. Biswal N. Nath 《Indian journal of clinical biochemistry : IJCB》1994,9(1):9-12
Membrane hydrophobicity and slalidase activity of normal Poly morphonuclear Leucocyte were significantly enhanced when incubated with DSF. As a consequence, internalisation ofE. coli andS. aureus (opsonised or unopsonised) were greatly dimnished, internalisation ofE. coli being higher in either category. Although, increase in hydrophobicity of the membrane correlated well with the time of decrease of particle internalisation (both at 30 min.), enhancement of sialidase activity did not coincide with the said alterations. 相似文献
12.
Uterine fluid, basically the endometrial secretion, is in dynamic exchange by influx and efflux with the oviducal and cervical fluids. During investigation the uterine fluid of both parous and IUCD women was collected and evaluated for inorganic phosphorus and cholesterol. The results of parous women were compared with the results of IUCD women. The trend of fluctuation in inorganic phosphorus concentration during different phases of menstrual cycle were same in both parous and IUCD women. But there was an increase in concentration in case of IUCD women in all stages of the menstrual cycle. After comparison with parous women a decrease in uterine fluid cholesterol concentration during proliferative period and increase during luteal period of IUCD women was observed. This increase in inorganic phosphorus and decrease and an increase in cholesterol concentration in IUCD women might be responsible in making uterine fluid environment unfavourable for sperm survival and blastocyst implantation. 相似文献
13.
Fuchs (2010, 2012) argues that users of social media produce value and surplus value in the Marxian sense. Arvidsson and Colleoni (2012) critique this hypothesis, claiming that Marx's theory of value is irrelevant to the regime of value production on social media platforms in particular and in informational capitalism in general. They claim that the affective relations and financial speculations that generate value on social media are not dependent on labor time. This article critically engages Fuchs, and Arvidsson and Colleoni, by revisiting Marx's theory of value. Contra Fuchs, we argue that audiences do not produce value and surplus value—neither for social nor for mass media. Contra Arvidsson and Colleoni, we argue that so-called affective relations (philia) do not produce value either. Instead we demonstrate that social media generate revenue from four primary sources—by leasing advertisement space to generate advertisement rent, by selling information, by selling services to advertisers, and by generating profits from fictitious capital and speculative windfalls. All four, we argue, can be adequately explained by Marx's theory of value. 相似文献
14.
Introduction:
Intensive exercising may significantly damage muscles which is reflected in pain, fatigue and the increase of muscle proteins concentrations in blood such are creatinin kinase (CK), lactic dehydrogenase (LD), myoglobin (MB) and other biochemical parameters including urea serum concentration (SU). Biochemical markers vary with age, sex, race, muscle mass, physical activity and climate conditions. They also assist us in determining the limit between the capacity for adaptation to given training process which results in supercomepensation and in condition of overtraining (OT), in the case of load that exceeds the physiologic potential of regeneration. Concerning the problem of diagnosis and explanation of the symptoms of overtraining, markers that can apply reliably and with sufficient sensitivity and simplicity of interpretation in the praxis are sought. It is critical to take into account difference among individuals and groups that could hamper the interpretation.The most frequently used markers:
The most frequently used biomarkers that provide us with the information on physical activity and on the amount of load through exercise are CK, SU and LD. Level of serum retaining kinas has been measured and interpreted for years as part of different scientific and professional investigations and presents one of basic parameters for determining the level of muscle damage. It reaches maximal concentration of the fourth day of exercising which depends on the type of exercise and the nature of stress triggered by exercise but also on individual characteristics.The level of serum urea presents marker of nitric compounds metabolism and is the principle chemical substance in the urine of mammals. It is thus possible to draw a parallel between the increases of serum urea concentration on increased degradations of proteins. Significant fall of serum amino acid levels occurs after sixty to seventy minutes of exercising with the increase of urea and free tyrosine and these changes have high correlation with the duration and intensity of.LD changes are important index of well-trained sportsmen and their capability to withstand the pace and force during strain in the training process. The level of LD is a good index of exercise intensity and marker of metabolic exchange in tissues whose concentration in serum is dependent of cell damage.Conclusion:
There is not a single, unique parameter that would provide enough valuable information for the estimation of the quality of exercising, amount of load and identification of overtraining. Delayed measurement of biomarkers is far from ideal, but it is obvious that the amount of stress/ load in training is the most important factor for the development of state of overtraining. Daily body weight control, diet, biochemical indices values and the input of water should be known and standardized before measurements. For the most of parameters determination of basal levels are needed in specific populations for more accurate interpretation and evaluation of results. The sampling process itself should be under the most strict conditions of standardization by repeating measurement at least every third day. Dependence of mentioned parameters (SU, CK, LD) on exercise intensity varies among individuals and without these additional measurements and subpopulation evaluations it is difficult to come to conclusions with certainty as well as to come to conclusions on causative correlations of training load and dynamic in biochemical parameters.Biochem Med (Zagreb) 2013 Jun; 23(2): A57–A58. Published online 2013 Jun 15. doi: 10.11613/BM.2013.027Common sports injuries
Miljenko FranićAuthor information Copyright and License information DisclaimerDubrava University Hospital, ZagrebCorresponding author: rh.dbk@cinarfm©Copyright by Croatian Society of Medical Biochemistry and Laboratory MedicineThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Sports injuries are injuries that occur in athletic activities and can be broadly classified as either traumatic or overuse injuries. Traumatic injuries because of the dynamic and high collision are nature of some sports. Overuse injuries cause wear and tear on the body, particularly on joints subjected to repeated activity.At every age, competitive and recreational athletes sustain a wide variety of soft tissue, bone, ligament, tendon and nerve injuries, caused by direct trauma or repetitive stress. Different sports are associated with different patterns and types of injuries, whereas age, gender and type of activity influence the prevalence of injuries. Sports trauma commonly affects joints of the extremities or the spine.The hip, knee and ankle are at risk of developing osteoarthritis (OA) after injury or in the presence of malalignment, especially in association with high impact sport. Spine pathologies are associated more commonly with certain sports. Upper extremity syndromes caused by a single stress or by repetitive micro-trauma occur in a variety of sports.Random control trials expose some subjects, but not others, to an intervention. This is more clinical in nature and not typically appropriate for the study of injury patterns. Cohort studies monitor both injured and non-injured athletes, thereby providing results on the effects of participation. Case-control studies monitor only those athletes who suffered an injury. The Ideal study would be Cohort design conducted over several teams, with longitudinal prospective data collection and one recorder where possible, as well as uniformity of injury definition across sports so comparisons between studies can be made accurately.Physical injury is an inherent risk in sports participation and, to a certain extent, must be considered an inevitable cost of athletic training and competition. Injury may lead to incomplete recovery and residual symptoms, drop out from sports, and can cause joint degeneration in the long term.Advances in arthroscopic techniques allow operative management of most intraarticular post-traumatic pathologies in the lower and upper limb joints, but long-term outcomes are not available yet. It is important to balance the negative effects of sports injuries with the many benefits that a serious commitment to sport brings.Biochem Med (Zagreb) 2013 Jun; 23(2): A58–A59. Published online 2013 Jun 15. doi: 10.11613/BM.2013.027Determination of sample size and number of study groups in sport studies
Mladen PetrovečkiAuthor information Copyright and License information DisclaimerDepartment of Laboratory Diagnosis, Dubrava University Hospital, Zagreb, Croatia, and Department of Medical Informatics, Rijeka University School of Medicine, RijekaCorresponding author: rh.irdem@pnedalm;©Copyright by Croatian Society of Medical Biochemistry and Laboratory MedicineThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 相似文献15.
Large-library fluorescent molecular arrays remain limited in sensitivity (1 × 106 molecules) and dynamic range due to background auto-fluorescence and scattering noise within a large (20–100 μm) fluorescent spot. We report an easily fabricated silica nano-cone array platform, with a detection limit of 100 molecules and a dynamic range that spans 6 decades, due to point (10 nm to 1 μm) illumination of preferentially absorbed tagged targets by singular scattering off wedged cones. Its fluorescent spot reaches diffraction-limited submicron dimensions, which are 104 times smaller in area than conventional microarrays, with comparable reduction in detection limit and amplification of dynamic range.Commercially available fluorescent micro-arrays based on target labeling, northern blot, or enzyme-linked immunosorbent assay (ELISA) are limited to a detection threshold of 1 to 10 × 106 molecules per fluorescent spot,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 thus requiring cell culturing or Polymerase Chain Reaction (PCR) amplification for many applications. The low sensitivity is often due to broad illumination, which creates auto-fluorescence noise. Even if point illumination and pin-hole filtering of non-focal plane noise are implemented in a confocal setup, the large and non-uniform fluorescent spots create scattering noise over each 20–100 μm element, which degrades the detection limit.4 Smaller spots can, in theory, be introduced by nano-sprays and nano-imprinting. However, directing the targets to such small areas then becomes problematic. Real-time PCR is, in principle, capable of detecting a single molecule but is limited in its target number5 and is hence slow/expensive for large-library assays. A large-library platform with much better detection limit than the current fluorescent microarrays would transform many screening assays. Ideally, this platform would not use the confocal configuration. Instead, it would direct the target molecules to a submicron spot and illuminate them with a nearby point source that does not require scanning.A promising platform is the optical fiber bundle array,6 with more than 104 fibers and targets, in principle. With its endoscopic configuration, these fiber bundles are most convenient for in situ and real-time biosensing modalities in microfluidic biochips and microfluidic 3-D cell cultures. Consequently, the optical sensing is typically carried out in the transmission mode, with the optical signals transmitted through the optical fibers to a detector. Microwell arrays at the distal end of imaging fiber, with molecular targets captured and transported to the microwells by microbeads, are the most popular among these optical fiber arrays. Although detection limit better than 1 × 106 molecules per bead has been reported, the bar-coded beads limit the target number of this platform.7, 8Our previous work9, 10 has shown that plasmonics at nanotips can enhance local electric field by three orders of magnitude. However, conduction loss and quenching of fluorescence11, 12 by the metal substrates limit the use of plasmonic enhanced fluorescence for large-library assays. Only nano-molar sensitivity has been demonstrated using plasmonics from metal coated nanocone tips.13, 14 In this paper, we will extend the conical fiber array platform not by tip plasmonics but by another optical phenomenon with induced dipoles: singular scattering off dielectric wedges and tips.15 Instead of the surface plasmon resonance on metallic nanostructures,16 field focusing at the cone tip by the dielectric media (the silica fiber) is used to produce a localized and singularly large scattering intensity at the tip. Singular scattering from a wedge or a cone has been known for decades.17, 18 It is only recently that numerical simulation19 has revealed that field focusing by this singular scattering can effect a five-order intensity enhancement that is frequency independent. This intense tip scattering produces a local light source at the tip that does not suffer from conduction loss. Unlike plasmonic metal nanostructures, the dielectric tip would also not quench the fluorescent reporters excited by the light source. In fact, it will help scatter the fluorescent signal, with Rayleigh scattering intensity scaling with respect to wavelength. We hence utilize this phenomenon for diffraction-limit fluorescent sensing/imaging for the first time here.The local light source due to tip scattering minimizes background auto-fluorescence and scattering noise, provided the target molecules preferentially diffuse towards the dielectric vertices. If the targets do not preferentially hybridize with probes at the vertices, there would be significant target loss, with a concomitant loss in sensitivity, because the vertex regions are just a small fraction of the total area. Fortunately, like electromagnetic radiation at the electrostatic limit of the Maxwell equations for sharp (sub-wavelength) vertices,20 the steady-state diffusion of molecules also obey the Laplace equation and so do the DC or AC electric potentials that drive electrophoresis and dielectrophoresis of the molecules.21 Hence, the diffusive, electrophoretic, and dielectrophoretic fluxes of target molecules are also singularly large at the vertices and there will be preferential hybridization there until the tip is saturated. Previously, we have demonstrated preferential diffusive transport of colloids to channel corners22 and dieletrophoretic trapping of bacteria23 and DNA molecules24 around sharp nanostructures like carbon nanotubes. Hence, dielectric nanotips fabricated by low-cost techniques can potentially provide the smallest fluorescent spot, which can preferentially capture target molecules and whose fluorescent image is limited in size only by the diffraction limit, without a confocal configuration.Although the scattering singularity is stronger at the conic tip, the total increase in scattering area of this singularity of measure zero is not as high as that of a sharp wedge, thus rendering the signal relatively weak. We hence employ a well-defined multi-wedged silica cone fabricated by wet-etching, with the wedges introduced by non-uniform stress formed during the fiber assembly process, to produce maximum scattering at the tip where three to four wedges converge (see inset of Fig. Fig.1A).1A). Using the reflection mode to fully exploit this singular scattering to excite fluorescent reporters at the tip and transmit the resulting signal, we report a nanocone array that can detect down to 100 molecules per cone tip with a large dynamic range from femtomolar to nanomolar concentrations. Although quantification for a single target is reported in this preliminary report, multi-target assays can readily be developed.Open in a separate windowFigure 1(A) A SEM image of the silica cone array where the single cone inset image shows three wedges converging into a 10 nm junction at the tip. (B) The optical setup of measurement. (C) The diffraction-limited fluorescent spot images.Amine-modified 35-base oligo-probes were functionalized onto both unetched silica fibers (as a control) and etched conic silica tips. The sample of 35-base ssDNA targets (corresponding to a primer for a segment of the Serotype 2 dengue genome) with a 5′ tagged Cy3 fluorophore was inserted into a microfluidic chip housing the fiber bundle (Fig. (Fig.1B)1B) and left overnight (see the supplementary material25 for exact sequence). After a standard rinsing protocol, fluorescent images were taken with an Olympus IX-71 fluorescent microscope for target concentrations ranging from 1 fM to 1 nM. A typical fluorescent image after hybridization is shown in Fig. Fig.1c,1c, where each micron-sized bright spot corresponds to a single tip in the cone array. The intensity profile shown in the supplementary material25 indicates a fluorescent spot smaller than 1 μm, indicating that the fluorescent light source is sub-wavelength and the resolution is close to diffraction limit. The size of this bright spot at the conic tip does not vary much with respect to the concentration but its intensity does, as shown in Fig. Fig.2A.2A. It was found that for flat fibers, only concentrations higher than 1 nM produced significant signals above the background. However, for etched conic fibers, 10 fM is clearly distinguishable from the background, which indicates that an improvement of sensitivity up to five orders can be realized by simply etching the flat surface into cone arrays. It also suggests very little target loss due to preferential hybridization onto the cone at sub-nM concentrations. We estimated the number of molecules per cone from the total number of molecules in target solution divided by the number of pixels on each fiber (104), which suggests less than 100 molecules per cone for a 10 fM bulk concentration, four orders better than any existing technology.Open in a separate windowFigure 2(A) Fluorescent intensity of etched conic fiber and unetched fiber for different concentrations of target molecules from 1 fM to 1 nM. (B) Fluorescent intensity increases linearly with exposure time. Non-target molecules with 1 μM concentration do not produce significant signal compared to lower concentrations of target molecules such as 1 nM and 10 nM (see the supplementary material25 for details of image analysis).Selectivity of the platform was also examined. Fig. Fig.2B2B presents the fluorescent intensity of the tips for non-target (1 μM) and target (1 nM and 10 nM) at different exposure times, which shows that fluorescent intensity increases linearly with exposure time. Beyond 5 s, saturation of images prevents further increase in the signal. For non-target, the intensity is much lower than 1 nM Target and 10 nM Target, which means non-target do not bind to the probes at the wedged tip as effectively as target molecules. Non-specific binding can be further removed by using more stringent buffers and higher flow rates.26 This platform can be extended to detect 70 000 targets, in theory, by functionalizing different probes onto each cones using localized photochemistry via masking, micro-mirror directed illumination, or direct laser writing. Extension to ELISA type protein assays is also straight forward. Integration of a transmission-mode optical fiber endoscope into a microfluidic biochip and into a 3-D cell culture for real-time monitoring of multiple molecular targets at near-single molecule resolution is currently underway. 相似文献
16.
Jaehoon Chung David Issadore Adeeti Ullal Kyungheon Lee Ralph Weissleder Hakho Lee 《Biomicrofluidics》2013,7(5)
We present a hybrid magnetic/size-sorting (HMSS) chip for isolation and molecular analyses of circulating tumor cells (CTCs). The chip employs both negative and positive cell selection in order to provide high throughput, unbiased CTC enrichment. Specifically, the system utilizes a self-assembled magnet to generate high magnetic forces and a weir-style structure for cell sorting. The resulting device thus can perform multiple functions, including magnetic depletion, size-selective cell capture, and on-chip molecular staining. With such capacities, the HMSS device allowed one-step CTC isolation and single cell detection from whole blood, tested with spiked cancer cells. The system further facilitated the study of individual CTCs for heterogeneity in molecular marker expression.Circulating tumor cells (CTCs) have emerged as an important biomarker in clinical practice as well as in fundamental research.1, 2 CTCs, shed from primary tumors, have been shown to be an early harbinger of tumor expansion and metastasis3 and have been used to predict disease progression, response to treatment, relapse, and overall survival.4, 5, 6 Recent work has shown that CTCs display distinct proteomic and genetic profiles; for example, CTCs in pancreatic cancer, have increased RNA expression of Wnt, implicating this pathway in metastasis.7 Proteomic characterization of proliferative markers such as Ki-67, and hormonal markers such as androgen receptor in prostate cancer, also have been shown to be predictive of treatment outcome.8, 9Despite such clinical potential of CTCs, their routine detection and characterization still remains a significant technical challenge.10 The task requires screening of a large number of cells (e.g., > 107 cells in 10 ml blood) and enrichment of heterogeneous targets against a complex biological background. Two main methods of CTC isolation are typically used: positive and negative selection. In positive selection, CTCs are directly isolated from blood via size-based filtration11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or antibody-based capture.1, 8, 21 Negative depletion reduces abundant blood cells, often by immunomagnetic separation, for downstream CTC enrichment.22 Both approaches have been used for high throughput CTC isolation from whole blood (SI Table 1).23 Each method, however, has its own inherent limitations. Positive enrichment could be biased by its selection criteria (e.g., cell size and cell surface markers). Negative selection, albeit unbiased, often requires complex sample processing (e.g., multiple washing steps for CTC isolation) that could result in cell loss.We hypothesized that both positive and negative selection could be combined in a single platform to enable (1) highly efficient and unbiased CTC purification, and (2) in-situ molecular analyses of collected cells. As a proof-of-concept, we herein describe a hybrid magnetic/size-sorting (HMSS) system that integrates magnetic and size-based isolation into a compact microfluidic chip. The HMSS first uses a magnetic filter to deplete leukocytes through immunomagnetic capture. Samples then pass through a size-sorter region that traps individual cells at predefined locations. Since abundant leukocytes are removed by the magnetic filter, the size-sorter could have a low size cut-off (∼5 μm), which allows for the unbiased capture of even small cancer cells. Furthermore, molecular probes can be introduced to perform on-chip, multiplexed analyses at single-cell resolution. We evaluated the utility of the developed system by capturing and profiling tumor cells in whole blood. The HMSS offers the advantages of both negative and positive selection and thereby differs from the recently reported iChip system24 which can operate only in either a negative or a positive selection mode. 相似文献
17.
We present a simple method for creating monodisperse emulsions with microfluidic devices. Unlike conventional approaches that require bulky pumps, control computers, and expertise with device physics to operate devices, our method requires only the microfluidic device and a hand-operated syringe. The fluids needed for the emulsion are loaded into the device inlets, while the syringe is used to create a vacuum at the device outlet; this sucks the fluids through the channels, generating the drops. By controlling the hydrodynamic resistances of the channels using hydrodynamic resistors and valves, we are able to control the properties of the drops. This provides a simple and highly portable method for creating monodisperse emulsions.Droplet-based microfluidic devices use micron-scale drops as “test tubes” for biological reactions.1, 2, 3 With the devices, the drops are loaded with cells, incubated to stimulate cell growth, picoinjected to introduce additional reagents, and sorted to extract rare specimens.4, 5, 6 This allows biological reactions to be performed with greatly enhanced speed and efficiency over conventional approaches: by reducing the drop volume, only picoliters of reagent are needed per reaction, while through the use of microfluidics, the reactions can be executed at rates exceeding hundreds of kilohertz. This combination of incredible speed and efficient reagent usage is attractive for a variety of applications in biology, particularly those that require high-throughput processing of reactions, including cell screening, directed evolution, and nucleic acid analysis.7, 8 The same advantages of speed and efficiency would also be beneficial for applications in the field, in which the amount of material available for testing is limited, and results are needed with short turnaround. However, a challenge to using these techniques in field applications is that the control systems developed to operate the devices are intended for use in the laboratory: to inject fluids, mechanical pumps are needed, while computers must adjust flow rates to maintain optimal conditions in the device.9, 10, 11, 12 In addition to significantly limiting the portability of the system, these qualities make them impractical for use outside the laboratory. For droplet-based microfluidic techniques to be useful for applications in the field, a general, robust, and portable system for operating them is needed.In this paper, we introduce a general, robust, and portable system for operating droplet-based microfluidic devices. In this system, which we call syringe-vacuum microfluidics (SVM), we load the reagents needed for the emulsion into the inlets of a microfluidic drop maker; using a standard plastic syringe, we generate a vacuum at the outlet of the drop maker,13 sucking the reagents through the channels, generating drops, and transporting them to different regions for visualization and analysis. By controlling the vacuum strength and channel resistances using hydrodynamic resistors14, 15, 16 and single-layer membrane valves,17, 18 we are able to specify the flow rates in different regions of the device and to adjust them in real time. No pumps, control computers, or electricity is needed for these operations, making the entire system portable and of potential use for field applications. To characterize the adjustability and precision of this system, we vary channel resistances and vacuum pressures while measuring the effects on drop size and production frequency. We also show how to use this to form drops of many distinct reagents simultaneously using only a single vacuum syringe.Monodisperse drop formation is the central operation in droplet-based microfluidics but can be quite challenging due to the need for precise, steady pumping of reagents; forming monodisperse drops with controlled properties is thus a stringent demonstration of the effectiveness of a control system. While there are many geometries available for microfluidic drop formation,19 in this discussion we use a simple cross-junction for its proven ability to form uniform emulsions at high rates of speed,20, 21 a schematic of which is shown in Fig. Fig.1.1. The devices are fabricated in poly(dimethylsiloxane) (PDMS) using soft lithography.22 The drop formation channels have dimensions of 25 μm in width and 25 μm in height. To enable production of aqueous drops in oil, which are the most useful for biological assays, we require hydrophobic devices, which we achieve using an Aquapel chemical treatment: we flow Aqualpel through the channels for a few seconds, flush with air, and then bake the devices for 20 min at 65 °C. After this treatment, the channels are permanently hydrophilic, as is needed for forming aqueous-in-oil emulsions. To introduce reagents into the device, we use 200 μl plastic pipette tips inserted into the channel inlets. To apply the suction, we use a 10 ml Bectin-Dickenson plastic syringe coupled to the device through a 16 G needle and PE∕5 tubing. The other end of the tubing is inserted into the outlet of the device.Open in a separate windowFigure 1Schematic of the microfluidic drop maker for use with SVM. To form water drops in oil, the device must be hydrophobic, which we achieve by treating the channels with Aquapel. The water and surfactant-containing oil are loaded into pipette tips inserted into the device inlets at the locations indicated. To pump the fluids through the drop maker, a syringe applies a vacuum to the outlet; this sucks the fluids through the drop maker, forming drops. The drops are collected into the suction syringe, where they can be stored, incubated, and reintroduced into a microfluidic device for additional processing.To begin forming drops, we fill the device with HFE-7500 fluorocarbon oil, displacing trapped air bubbles that could restrict flow and interfere with drop formation. Pipette tips containing reagents are then inserted into the device inlets, as shown in Fig. Fig.11 and pictured in Fig. Fig.2a;2a; during this step, care must be taken to not trap air bubbles under the pipette tips, as they would restrict flow. For the fluids, we use distilled water for the droplet phase and HFE-7500 with the ammonium salt of Krytox 157 FSL at 1.8 wt % for the continuous phase. The suction syringe is then connected to the device outlet; to initiate drop formation, the piston is pulled outward and locked in place with a 1 in. binder clip, as shown in Fig. Fig.2a.2a. This expands the air in the syringe, generating a vacuum that is transferred to the device through tubing. Since the inlet reagents are open to the atmosphere and thus maintained at a pressure of 1 atm, this creates a pressure differential through the device that pumps the fluids. As the fluids flow through the cross-channel, forces are generated that create drops, as shown in Fig. Fig.2b2b (enhanced online). Due to the very steady flow, the drops are highly monodisperse, as shown in Fig. Fig.2c.2c. After they are formed, the drops flow out of the device through the suction tube and are collected into the syringe. Depending on the emulsion formulation, drops may coalesce on the metal needle of the syringe; if so, an Upchurch fitting should be used to couple the tubing instead. The collected drops can be stored in the syringe, incubated, and reintroduced into additional microfluidic devices, as needed for the assay.Open in a separate windowFigure 2Photograph of the microfluidic drop formation device with pipette tips containing emulsion reagents and vacuum syringe for pumping (a). Distilled water is used for the droplet phase and HFE-7500 fluorocarbon oil with fluorinated surfactant for the continuous phase. The vacuum applies a pressure differential through the device that pumps the fluids through the drop maker (b) forming drops. The drops are monodisperse, due to the controlled properties of drop formation in microfluidics (c). The scale bars denote 50 μm (enhanced online).In many biological applications, drop size must be precisely controlled. This is essential, for example, when encapsulating molecules or cells in the drops, in which the number encapsulated depends on the drop size.3, 23, 24 With SVM, the drop size can be precisely controlled. Our strategy to accomplish this is motivated by the physics of microfluidic drop formation. In microfluidic devices, the capillary number of the flow is normally small, Ca<0.1; as a consequence, the drop formation physics follows a plugging∕squeezing mechanism, in which the drop size depends on the flow rate ratio of the dispersed-to-continuous phase.20, 25 By adjusting this ratio, we can thus control the drop size. To adjust this ratio, we use hydrodynamic resistor channels.14, 15, 16 These channels are analogous to electronic resistors in that for a fixed pressure drop (voltage) the flow rate through them (current) is inversely proportional to their resistance. By making the resistors longer or shorter, we adjust their resistance, thereby controlling the flow rate.To use resistors to control the drop size, we place three on the inlets of the cross-junction, at the locations indicated in Fig. Fig.3a.3a. In this configuration, the flow rate ratio depends on the resistances of the central and side resistors: shortening the side resistors increases the continuous phase flow rate with respect to the dispersed phase, thereby reducing the ratio and, consequently, the drop size, whereas lengthening it increases the drop size. By varying the ratio, we produce drops over a range of sizes, as shown in Fig. Fig.3b3b (enhanced online). The drop size is linear in the resistance ratio, indicating that it is linear in the flow rate ratio, as is expected for plugging∕squeezing drop formation [Fig. [Fig.3b3b].20, 25 This behavior is identical to that of pump-driven fluidics, demonstrating that SVM affords similar control.Open in a separate windowFigure 3Drop properties can be controlled using resistor channels. The resistors are placed on the inlets of the drop maker at the locations indicated in (a). The resistors enable the flow rates of the inner and continuous phases to be controlled. By varying the length ratio of the inlet resistors, we control the flow rate ratio in the drop maker. This allows the drop volume to be controlled, as shown by drop volume plotted as a function of inlet resistor length ratio in (b); varying this ratio does not significantly affect the drop formation frequency, as shown in (c). By varying the length of the outlet resistor, we control the total flow rate through the device; this allows us to form drops of constant volume, but at a different formation frequency, as shown by the plots of volume and frequency as a function of the inverse of the outlet resistor length in (d) and (e), respectively. The measured hydrodynamic resistance of a resistor channel with water as a function of length is shown as inset into (d) (enhanced online).We can also control the frequency of the drop formation using resistor channels. We place a resistor on the outlet of the device; this sets the total flow rate through the device, thereby adjusting drop frequency, as shown in Fig. Fig.3e3e (enhanced online). To confirm that the size and frequency control are independent, we plot size as a function of the outlet resistance and frequency as a function of the resistance ratio [Figs. [Figs.3c,3c, ,3d];3d]; both are constant as a function of these parameters, again demonstrating independent control. Frequency can also be adjusted by changing the strength of the vacuum, which can be accomplished by loading a prescribed volume of air into the syringe before expansion. In this case, the vacuum pressure applied is Pfin=Vin∕Vfin×Pin, where Vin is the initial volume of air in the syringe, Vfin is the volume after expansion, and Pin is the initial pressure, which is 1 atm. By loading a prescribed volume of air into the syringe before connecting it to the device and pulling the piston, the expansion factor can be reduced, thereby lowering the vacuum strength.The flow rates through the microfluidic device depend on the applied pressure differential, which, in turn, depends on the value of the ambient pressure. Since ambient pressure may vary due to differences in altitude, the drop formation may also vary. However, since ambient pressure variations affect the inner and outer phase flows equally, this should alter the total flow rate but not the flow rate ratio. Consequently, we expect it to alter drop formation frequency but not drop size because while the frequency depends on absolute flow rate [as illustrated by Fig. Fig.3e],3e], drop size depends on the flow rate ratio [as illustrated in Fig. Fig.3b].3b]. Based on normal variations in atmospheric pressure on the surface of the Earth, we expect this to produce differences in the drop formation frequency of ∼25%, for example, when operating a device at sea level compared to at the top of a moderately sized mountain.Resistor channels allow drop properties to be controlled, equivalent to what is possible with pump-driven flow; however, they do not allow real-time control because their dimensions are fixed during the fabrication. Real-time control is often needed, for example, as it is when performing reactions in drops for the first time, in which the optimal drop size is not known. To enable real-time control, we must adjust flow rates, which can be achieved using the fluidic analog of electronic potentiometers. Single-layer membrane valves are analogous fluidic components, consisting of a control channel that abuts a flow channel.17, 18 By pressurizing the control channel, the thin PDMS membrane between these channels is deflected laterally, constricting the flow channel, thereby increasing its hydrodynamic resistance and reducing its flow rate.18 To use these membrane valves to vary drop size, we replace the inlet resistors with inlet valves, as shown in Fig. Fig.4a.4a. To set the flow rate through a path, we actuate the valve with a defined pressure. To actuate the valves, we use air-filled syringes: a 1 ml syringe is filled with air and connected to the valve control channel through tubing; an additional component, a three-way stopcock is inserted between the syringe and needle, allowing the pressure to be locked in after optimal actuation conditions are obtained. We use one syringe to control the dispersed phase valves and another to control the continuous phase valves. The valves are pressurized by compressing the air in the syringes to a defined degree using the marked graduations; this is achieved by pressing the piston to a defined graduation mark, compressing the air contained within it, thus increasing pressure. The stopcock is then switched to the off position, locking in the actuation. This simple scheme allows precise actuation of the valves, for accurate, defined flow rates in the drop maker, and controlled drop size, as shown in Figs. Figs.4b,4b, ,4c4c (enhanced online). The drop size can be varied at a rate of several hertz without noticeable loss of control; moreover, changing the drop size does not affect the frequency, indicating that, again, these properties are independent, as shown by the constant drop frequency with varying pressure ratio in Fig. Fig.4d4d.Open in a separate windowFigure 4Single-layer membrane valves allow the drop size to be varied in real time to screen for optimal reaction conditions. The valves are positioned on the inner and side inlets, as indicated in (a). By adjusting the actuation pressures of the valves, we vary the flow rates in the drop maker, thereby changing the drop size (b), as shown by the plot of drop volume as a function of the actuation pressure ratio in (c). Varying the inlet resistance ratio does not significantly alter drop formation frequency, as shown by frequency as a function of the pressure ratio in (d). A movie of drop formation during actuation of the valves are available in the supplemental material (Ref. 29). The scale bars denote 100 μm (enhanced online).Another useful attribute of SVM is that it readily lends itself to parallel drop formation26 because the pressure that pumps the fluids through the channels is supplied by the atmosphere and is applied evenly over the whole outer surface of the device. This allows fluids to be introduced at equal pressures from different inlets, for forming drops with identical properties in different drop makers. To illustrate this, we use a parallel drop formation device to emulsify eight distinct reagents simultaneously; the product of this is an emulsion library, consisting of drops of identical size in which different drops encapsulate distinct reagents, useful for certain biological applications of droplet-based microfluidics.7 The microfluidic device consists of eight T-junction drop makers.25 The drop makers share one oil inlet and outlet but each has its own inner-phase inlet, as shown in Fig. Fig.5.5. The oil and outlet channels are wide, ensuring negligible pressure drop through them, so that all T-junctions are operated under the same flow conditions. A distinct reagent fluid is introduced into the inner phase of each T-junction, for which we use eight concentrations of the dye Alexa Fluor 680 in water. After loading these solutions into the device through pipette tips, a syringe applies the vacuum to the outlet, sucking the reagents through the T-junctions, forming drops, as shown by the magnified images of the T-junctions during drop formation in Fig. Fig.5.5. Since the drop makers are identical and operated under the same flow conditions, the drops formed are of the same size, as shown in the magnified images in Fig. Fig.55 and in a movie available in the supplemental material.29Open in a separate windowFigure 5Parallel drop formation device consisting of eight T-junction drop makers. The drop makers share a common oil inlet and outlet, both of which are wide to ensure even pressure distribution to all drop makers; support posts prevent these channels from collapsing under the suction. Each drop maker has its own inner-phase inlet, allowing emulsification of a distinct reagent. Since the drop maker dimensions and pressure differentials are constant through all drop makers, the drops formed are of the same size, as shown in the magnified images. The drops are ∼35 μm in diameter.To verify that the dye solutions are successfully encapsulated, we image a sample of the collected drops with a fluorescent microscope. The drops are confined in a monolayer between two glass plates so they can be individually imaged. They are of the same size but have distinct fluorescence intensities, as shown in Fig. Fig.6a.6a. To quantify these differences, we measure the intensity of each drop and plot the results as a histogram [see Fig. Fig.6b].6b]. There are eight peaks in the histogram, corresponding to the eight dye concentrations, demonstrating that all dyes are encapsulated successfully. The peak areas are also similar, demonstrating that drops of different types are formed in equal amounts due to the uniformity of the parallel drop formation.Open in a separate windowFigure 6Fluorescent microscope image of emulsion library created with parallel T-junction device (a). In this demonstration, eight concentrations of Alexa Fluor 680 dye are emulsified simultaneously, producing an emulsion library of eight elements. The drops are of the same size but encapsulate distinct concentrations of the dye solution, as demonstrated by the eight peaks in the intensity histograms in (b). The scale bar denotes 100 μm.SVM is a simple, accessible, and highly controlled way to form monodisperse emulsions for biological assays. It allows controlled amounts of different reagents to be encapsulated in individual drops, drop size to be precisely controlled, and the ability to form drops of different reagents at the same time, in a parallel drop formation device. These properties should make SVM useful for biological applications of monodisperse emulsions;1, 2, 3 the portability of SVM should also make it useful for applications in the field, particularly when no electrical power source is available. The parallel emulsification technique should also be useful for particle templating from drops, in which the particles must be of the same size but composed of distinct materials.26, 27, 28, 29 相似文献
18.
Plasmonic hot spots, generated by controlled 20-nm Au nanoparticle (NP) assembly, are shown to suppress fluorescent quenching effects of metal NPs, such that hair-pin FRET (Fluorescence resonance energy transfer) probes can achieve label-free ultra-sensitive quantification. The micron-sized assembly is a result of intense induced NP dipoles by focused electric fields through conic nanocapillaries. The efficient NP aggregate antenna and the voltage-tunable NP spacing for optimizing hot spot intensity endow ultra-sensitivity and large dynamic range (fM to pM). The large shear forces during assembly allow high selectivity (2-mismatch discrimination) and rapid detection (15 min) for a DNA mimic of microRNA.Irregular expressions of a panel of microRNAs (miRNA) in blood and other physiological fluids may allow early diagnosis of many diseases, including cancer and cardiovascular diseases.1 However, quantifying all relevant miRNAs (out of 1000), with similar sequences over 22 bases2 and large variations in expression level (as much as 100 fold) at small copy numbers, requires a new molecular diagnostic platform with high-sensitivity, high-selectivity, and large dynamic range. Current techniques for miRNA profiling, such as Northern blotting,3 microarray-based hybridization,4 and real-time quantitative polymerase chain reaction5 are expensive and complex. A simple and rapid miRNA array would allow broad distribution of molecular diagnostic devices for cancer and chronic diseases, eventually into homes for frequent prescreening of many diseases.At their low concentrations in untreated samples, optical sensing of miRNA is most promising. Plasmonically excited Raman scattering (SERS) and fluorescence sensors from metallic nanoparticles (NPs) or surfaces have enhanced the sensitivity of optical molecular sensors by orders of magnitude.6, 7, 8, 9 However, probe-less SERS sensing or fluorescent sensing of unlabeled targets are insufficiently specific for miRNA targets in heterogeneous samples. Plasmonic detection is also very compatible with FRET probes whose donor dye offers small light sources to excite fluorescently labelled targets upon hybridization.7, 10A particular family of FRET reporters does offer label-free sensing: hairpin oligo probes whose end-tagged fluorophores are quenched by the Au NP to which they are functionalized.11 The fluorescent signal is only detected when the hairpin is broken by the hybridizing target nucleic acid or protein (for an aptamer probe), and the more rigid paired segment separates the end fluorophore from the quenching surface to produce a fluorescent signal. It is often hoped that plasmonics on the metal surface will enhance the intensity to overcome the quenching effect, if the linearized hairpin is within the NP plasmonic penetration length. However, since fluorescent quenching decays slowly (linearly) with fluorophore-metal spacing10 whereas the plasmonic intensity decays exponentially from a flat surface, careful experimentation shows that quenching dominates and the hairpin probe actually produces a larger intensity on non-metallic surfaces,10 on which it can not function as a label-free probe. Hence, only μM limit-of-detection (LOD) has been achieved with this technique on single NPs or on flat metal surfaces,12 with expensive laser excitation and confocal detection.Plamonic hot spots formed between metal nanostructures and sharp nanocones can further amplify the plasmonic field.13, 14 The hot spot intensity decays algebraically with respect to the separation or cone tip distance and hence should dominate the linear decay of the metal quenching effect at some optimum separation.15 It is hence possible that plasmonic hot spots may allow much lower LOD with inexpensive optical instruments—ideally light-emitting diode light source and miniature camera. However, the dimension of the gaps, cones, and wedges needs to be at nanoscale, and the cost is now transferred to fabrication of such hot-spot substrates like bow-ties, double crescents, bull-eyes, etc.16 Low-cost wet-etching techniques for addressable nanocones that sustain converging plasmonic hot spots17 have been reported but the fabricated nanocones are often too non-uniform to allow precise quantification. NP monolayers have been shown to exhibit plasmonic hot spots and fluorescence enhancement.18, 19 However, the enhancement only occurs within a range of spacing between aggregated NPs, which is difficult to control and the location or even the existence of the hotspots are not known a priori.Higher sensitivity is expected if a minimum number of NPs are used in an assembly at a known location and if the NP assembly can produce crystal-like aggregates with controllable NP spacing. Induced DC and AC NP dipoles (related to dielectrophoresis) have been used to assemble NP crystals by embedded micro-electrodes to provide the requisite high field.20, 21 The resulting NP crystals are ideal for plasmonic hot spots, since the spacing of the regimented NP crystal can be controlled by the applied voltage. Conic nanocapillaries22, 23 will be used here for such field-induced NP assembly because the submicron-tip can focus the electric field into sufficient high intensity for NP assembly without embedded-electrodes. Because the field is highest at the tip due to field focusing, the micron-sized crystal would be confined to a small volume, which will be shown to be less than typical confocal volumes, at a known location. So long as the hotspots are regimented, the quantification of target molecules is determined by the total fluorescent intensity and is hence insensitive to the exact geometry of the nanocapillary.Fluorescent microscope equipped with tungsten lamp light source and normal CCD camera from Q Imaging were used for simultaneous optical and ion current measurements, as shown in Fig. Fig.1a.1a. The nanocapillaries were pulled from commercial glass capillaries using laser-assisted capillary puller. SEM image of a typical pulled glass nanocapillary in Fig. Fig.1b1b shows an inner diameter of 111 nm and cone angle of 7.3°. The capillary was inserted into a Polydimethylsiloxane chip with two reservoirs. The 20 nm Au NPs, functionalized with fluorescently labelled dsDNA, were injected into the base reservoir. With SEM imaging (Fig. S3 in the supplementary material24), the functionalized DNA is found to prevent NP aggregation even in high ionic-strength Phosphate buffered saline buffer. The NP solution is then driven into the capillary through the tip by applying a positive voltage. Fig. Fig.1c1c shows the ion current evolution over 2 h at +1 V packing voltage. The ion current increases rapidly in the first 10 min, then at a much slower rate. The rise of current indicates assembly of conductive Au NP assembly at the tip. This was confirmed by the strong fluorescence signal at the tip region during the packing process (inset of Fig. Fig.1c).1c). The one-micron region (corresponding to roughly an aggregate volume of one attoliter) near the capillary tip shows a fluorescence signal after 1 min and also appeared as a dark spot in the transmission image (supplementary material, Fig. S124). This spot darkens with longer packing time but does not grow in size, consistent with the monotonically increasing ion current with increased packing density of the NP assembly. As contrast, a strong fluorescence appeared after only 1 min of packing, but the signal became weaker after 15 min (supplementary material, Fig. S124). This reduction in fluorescence is not due to bleaching of fluorophores because we took 2 images in 15 min at 5 s exposure each and control experiments show significant bleaching only beyond an exposure time of 100 s (see supplementary material).24 Instead, the non-monotonic dependence of the fluorescence intensity with respect to time is because of the optimal hotspot spacing for highest plasmonic intensity at about 5–20 nm,25, 26, 27 which is reached at about 10 min.Open in a separate windowFigure 1Plasmonic hotspots generated at the tip of a nano-capillary. (a) Schematic of the experimental set up. (b) SEM image of glass nanocapillary shows opening at the tip with a diameter of 111 nm. (c) Current evolution during packing of fluorescently labeled gold particles at +1 V. Inset shows strong fluorescence only after 1 min of packing.The FRET probe is designed to exploit the plasmonic hotspot.24 We first electrophoretically drove the target molecules in the tip side reservoir into the nano-capillary by applying a negative voltage of −1 V. During this process, the targets are trapped within the capillary and hybridize with the hairpin probes on the Au NP in the nanocapillary. Fluorescence of the unquenched hybridized probes is too weak to be detected by our detector as shown in Fig. Fig.2b.2b. A reverse positive voltage of +1 V was then applied to the capillary to pack the Au NPs to the tip. Due to plasmonic hot spots of aggregated gold nanoparticles, the fluorescence signal is significantly enhanced at the tip and can be detected by our CCD camera, as shown in Fig. Fig.2c2c.Open in a separate windowFigure 2(a) Schematics of designed hairpin probe on gold particle. (b) Before packing gold particles, probe fluorescence signal was too weak to be detect. (c) After packing for 3 minutes, a strong fluorescence signal appears at the NP aggregate. (d) Normalized intensity (average of all pixels above a threshold (15 au) normalized with respect to the average over all pixels (with 0-250 au)) as a function of packing voltage for different samples. Black, 1 nM target ; blue, 10 pM target; purple, 10 nM 2-mismatch non-target. (e) Intensity dependence on target concentration. Measured normalized intensity before packing (black) and after packing (red), for three independent experiments with different nano-capillaries at each concentration. NT stands for non-target at 10 nM as a reference.For the same packing time, the fluorescence intensity increases initially but saturates after 10 min time of trapping (supplementary material, Fig. S2(a)24). Over 10 min of trapping with a negative voltage, we found the fluorescence intensity exhibits a maximum at a packing time of 3 min (supplementary material, Fig. S2(b)24). In later experiments, we used 10 min trapping time and 3 min packing time as standards.Fig. Fig.2d2d shows the fluorescence intensity is sensitive to the positive packing voltage at different concentration of target and non-target molecules. For target samples (1 nM and 10pM), the optimal voltage is about 1 V. We suspect that with larger voltage, the NPs are packed too tightly such that the NP spacing is smaller than the optimal distance for plasmonic hotspots. The fluorescence intensity for a nontarget with two mismatches is 7 times lower than the target even with a 10 times higher concentration (10 nM). Moreover, the optimal voltage for the non-target miRNA is reduced to 0.5 V instead 1 V for the target miRNA. Strong shear during electrophoretic packing has probably endowed this high selectivity.20Using the protocol above, the LOD and dynamic range of the target was determined (Fig. (Fig.2e).2e). The intensity at each concentration is measured with three independent experiments with different nanocapillaries to verify insensitivity with respect to the nanocapillary. The intensity increases monotonically with respect to the concentration from 1fM to 1pM. Beyond 1pM, the fluorescence signal saturates, presumably because all hairpin probes at the tip have been hybridized. At 1 fM, the fluorescent intensity is still well above the background measured from the non-target sample. Note both auto-fluorescence of gold nanoparticles and free diffusing non-target DNA molecules contribute to the background. Given the volume of tip side reservoir (∼50 μl), there are about 30 000 target molecules in the reservoir at 1 fM. However, with a short 10 min trapping time, we estimate only a small fraction of these molecules, less than 100, have been transferred from the tip reservoir into the nanocapillary. 相似文献
19.
Wang Zhao Li Zhang Wenwen Jing Sixiu Liu Hiroshi Tachibana Xunjia Cheng Guodong Sui 《Biomicrofluidics》2013,7(1)
A microfluidic device was successfully fabricated for the rapid serodiagnosis of amebiasis. A micro bead-based immunoassay was fabricated within integrated microfluidic chip to detect the antibody to Entamoeba histolytica in serum samples. In this assay, a recombinant fragment of C terminus of intermediate subunit of galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica (C-Igl, aa 603-1088) has been utilized instead of the crude antigen. This device was validated with serum samples from patients with amebiasis and showed great sensitivity. The serodiagnosis can be completed within 20 min with 2 μl sample consumption. The device can be applied for the rapid and cheap diagnosis of other infectious disease, especially for the developing countries with very limited medical facilities.Entamoeba histolytica is the causative agent of amebiasis and is globally considered a leading parasitic cause of human mortality.1 It has been estimated that 50 × 106 people develop invasive disease such as amebic dysentery and amebic liver abscess, resulting in 100 000 deaths per annum.2, 3 High sensitive diagnosis method for early stage amebiasis is quite critical to prevent and cure this disease. To date, various serological tests have been used for the immune diagnosis of amebiasis, such as the indirect fluorescent antibody test (IFA) and enzyme-linked immunosorbent assay (ELISA).We have recently identified a 150-kDa surface antigen of E. histolytica as an intermediate subunit (Igl) of galactose and N-acetyl-D-galactosamine-inhibitable lectin.4, 5 In particular, it has been shown that the C-terminus of Igl (C-Igl, aa 603-1088) was an especially useful antigen for the serodiagnosis of amebiasis. ELISA using C-Igl is more specific than the traditional ELISA using crude antigen.6 However, the ELISA process usually takes several hours, which is still labor-intensive and requires experienced operators to perform. More economic and convenient filed diagnosis methods are still in need, especially for the developing countries with limited medical facilities.Among all the bioanalytical techniques, microfluidics has been attracting more and more attention because of its low reagent/power consumption, the rapid analysis speed as well as easy automation.7, 8, 9, 10, 11 Especially with the development of the fabrication technique, microfluidics chip can include valves, mixers, pumps, heating devices, and even micro sensors, so many traditional bioanalytical methods can be performed in the microfluidics. Qualitative and quantitative immune analysis on the microfluidic chip was successfully proved by plenty of research with improved sensitivity, shorten reaction time, and less sample consumption.8, 10, 11, 12, 13, 14, 15, 16, 17 Moreover, with the intervention of other physical, chemical, biology, and electronic technology, microfluidic technique has been successfully utilized in protein crystallization, protein and gene analysis, cell capture and culturing and analysis as well as in the rapid and quantitative detection of microbes.13, 14, 15, 16, 17, 18, 19, 20Herein, we report a new integrated microfluidic device, which is capable of rapid serodiagnosis of amebiasis with little sample consumption. The microfluidic device was fabricated from polydimethysiloxane (PDMS) following standard soft lithography.21, 22 The device was composed of two layers (shown in Figure Figure1)1) including upper fluidic layer (in green and blue) and bottom control layer (in red).Open in a separate windowFigure 1Structure illustration of microfluidic chip.To create the fluidic layer and the control layer, two different molds with different patterns have fabricated by photolithographic processes. The mold to create the fluidic channels was made by positive photoresist (AZ-50 XT), while the control pneumatic mold was made by negative photoresist (SU8 2025). For the chip fabrication, the fluidic layer is made from PDMS (RTV 615 A: B in ratio 5:1), and the pattern was transferred from the respective mold. The control layer is made from PDMS (RTV 615 A:B in ratio 20:1). The two layers were assembled and bonded together accurately, and there is elastic PDMS membrane about 30 μm thick between the fluidic layer channels and control layer.21, 22 The elastic membrane at the intersection can deform to block the fluid inside the fluidic channels, functioning as valves under the pressures introduced though control channels. There are two types of channels in fluidic layer, the rectangular profiled (in green, 200 μm wide, 35 μm thick) channel and round profiled channels (in blue, 200 μm wide, 25 μm center height). Because of the position of the valves on the fluidic channels, two types of valves (Figure (Figure2a)2a) were built, working as a standard valve and a sieve valve. The standard valves (on blue fluidic channels) can totally block the fluid because of the round profile of fluidic channel; the sieve valve can only half close because of the rectangular profile. The sieve valve can be used to trap the microspheres (beads) filled inside the green fluidic channels, while letting the fluid pass through. By this sieve valve, a micro column (in green) is constructed, where the entire ELISA reaction happens. The micrograph of the fabricated micro device is shown in Figure Figure2b.2b. The channels were filled with food dyes in different colors to show the relative positions of the channels. The pressures though different control channels are individually controlled by solenoid valves, connected to a computer through relay board. By programming the status (on/off) of various valves at different time periods, all the microfluidic chip operation can be digitally controlled by the computer in manual, semi-automatic, or automatic manner.Open in a separate windowFigure 2(a) Structure illustration of micro column, standard valve and sieve valve; (b) photograph of the microfluidic chip.To validate this device, 12 patient serum samples were collected. Sera from 9 patients (Nos. 1–9) with an amebic liver abscess or amebic colitis were used as symptomatic cases. The diagnosis of these patients was based on their clinical symptoms, ultrasound examination (liver abscess) and endoscopic or microscopic examination (colitis). We also identified the clinical samples using PCR amplification of rRNA genes.24 As negative control, sera obtained from 3 healthy individuals with no known history of amebiasis were mixed into pool sera. The serum was positive for E. histolytica with a titer of 1:64 (borderline positive), as determined by an indirect fluorescent-antibody (IFA) test.23, 24 In our previously study, the sensitivity and specificity of the recombinant C-Igl in the ELISA were 97% and 99%.6, 25 In the current study, the serodiagnosis of amebiasis was also examined by ELISA using C-Igl.26 The cut-off for a positive result was defined as an ELISA value > 3 SD above the mean for healthy negative controls27 (shown in Figure Figure3).3). The seropositivity to C-Igl was 100% in patients with amebiasis.Open in a separate windowFigure 3ELISA reactivity of sera from patients against C-Igl. ELISA plate was coated with 100 ng per well of C-Igl. Serum samples from patients and healthy controls were used at 1:400 dilutions. The dashed line indicates the cut-off value. Data are representative of results from three independent experiments.In the diagnosis process with microfluidic chip, the 4 micro immuno-columns filled with C-Igl-coated microspheres were the key components of the device. The C-Igl was prepared in E. coli as inclusion bodies. After expression, the recombinant protein was purified and analyzed by SDS-PAGE. The apparent molecular mass was 85 kDa.26The immune-reaction mechanism is illustrated in Figure Figure4.4. The anti-His monocolonal antibody was immobilized onto the microspheres (beads, 9 μm diameter) coated with protein A. The C-Igl was then immobilized onto the beads through the binding between the His tag and C-Igl. For the diagnosis, the microspheres immobilized with C-Igl and blocked by 5% BSA were preloaded into the columns for the rapid analysis of the patient serum samples. Generally, serum samples which were diluted 100 times were first loaded into the reaction column and incubated at room temperature for 5 min. After being washed by PBS buffer, FITC-conjugated goat anti-human polyclonal antibody was added into the column for 4 min incubation. The fluorescence image can be collected by the fluorescence microscope after the micro column was washed with PBS buffer. From loading diluted serum samples into column to collecting fluorescence images, the total time to complete the immunoassay is less than 10 min. The final fluorescence results were analyzed by Image Pro Plus 6.0.Open in a separate windowFigure 4Schematic representation of the ELISA in the chip.Different reaction conditions have been investigated to find the optimized ones. For each patient, 2 μl sample is enough for the analysis. The designed microfluidic chip with 4 micro columns is capable for 4 parallel analyses at the same time. More micro columns can be integrated into the device if more parallel tests are needed.Different incubating time for the diagnosis has also been investigated and no significant difference has been found for various time periods. It is enough to incubate the chip for only 5 min. The total diagnosis time for one sample is less than 10 min. The detection result appeared as the fluorescence intensity of the reaction column. As shown in Figure Figure5,5, the negative sample showed relatively low fluorescence intensity, because little FITC-conjugated goat anti-human polyclonal antibody could attach to the surface of microspheres; on the contrast, the positive sample showed much brighter fluorescence. The fluorescence intensity can be transferred to digital data (Table Sample Average scores Standard deviation 1 33 790 368 2 23 269 271 3 39 598 307 4 7784 52 5 21 222 197 6 38 878 290 7 22 437 227 8 36 295 334 9 41 024 396 Negative 200 32