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Cerebrospinal fluid (CSF) leakage due to incidental durotomy is an inherent complication of spine surgery. With appropriate treatment, complications of CSF leakage, such as headache and even meningitis, can be reduced. CSF leakage could be detected on the basis of correlated clinical symptoms; diagnosis should be based on these symptoms and appropriate imaging studies. However, the diagnosis of CSF leakage remains a challenge, especially if incidental durotomy is unrecognized during surgery; even if incidental durotomy is detected and repaired intraoperatively, the severity of the leakage and quality of the primary dural repair are difficult to evaluate postoperatively. Rapid, inexpensive, and safe methods of detecting CSF-containing samples are currently lacking; hence, the development of a point-of-care test (POCT) method to improve diagnostic efficiency is necessary. We developed a high-sensitivity lateral flow immunoassay with a stacking pad (sLFIA) for quantitative detection of β-trace protein (BTP), a specific CSF marker. The BTP concentration in 39 clinical samples was calculated using a calibration equation for test-line intensity and evaluated by a standard laboratory method. To avoid the hook effect, we diluted each sample prior to testing. The correlation coefficient between the enzyme-linked immunosorbent assay and our BTP sLFIA method was 0.991 A 75-fold sample dilution was applied owing to the hook effect point, identified as 175 ng mL

Tuberculosis is one of devastating infectious diseases in the world, and early diagnosis and treatment can help overcome this global burden. In this work, a new detection platform combining smartphone-assisted fluorescent analysis and highly sensitive fluorescent copper nanoprobes (CuNPs) in a specific nucleic acid amplification test (NAAT) for the diagnosis of tuberculosis (TB) was demonstrated and validated using clinical samples. To enhance the precision and accuracy of detection, polymerase chain reaction (PCR), padlock probe (PLP) ligation, and rolling circle amplification (RCA) were combined. Long poly(thymine) (polyT) single-stranded DNA was synthesized through RCA, and polyT-CuNPs were formed by adding copper(II) ions and sodium ascorbate as reducing agents; subsequently, the results were visualized through the excitation from a UV transilluminator and quantified with just a smartphone. After optimization, this proposed platform was validated by testing 18 residual DNA samples after TB PCR, including 8 TB-negative and 10 TB-positive samples, and exhibited a detection limit of 5 fg/μL. The findings indicate the potential of this platform for practical application, where it can be combined with a smartphone for image analysis to achieve accurate on-site detection of TB, especially in resource-limited settings.

Lateral flow immunoassays (LFIAs) have wide application in point-of-care testing, particularly in resource-poor settings. To achieve signal amplification in a gold nanoparticle-based lateral flow assay without an additional procedure or the need for complex fabrication, a new and simple method was developed for using a “stacking pad” configuration that adds an additional membrane between the conjugation pad and test pad to the conventional AuNP-based LFIA format. This design helps to accumulate the antibody and antigen on the stacking pad, hence extending the antigen/antibody binding interactions to enhance the test’s detection sensitivity. With the enhanced lateral flow assay, as low as 1 ng/mL of Protein A and 15.5 ng/mL of C-reactive protein can be visualized with the naked eye. We also successfully applied the stacking pad system in the analysis of C-reactive protein in human serum and synovial fluid samples. These results suggest that this stacking pad LFIA can provide sensitive and on-site prognosis for detection in synovial fluid and serum samples in resource-limited settings.

In this study, we investigated the antibacterial activity of silver-coated gold nanoparticles (Au-Ag NPs) immobilized on cellulose paper. Ag NPs are known to have strong antibacterial properties, while Au NPs are biocompatible and relatively simple to prepare. We made the Au-Ag NPs using a facile process called Ag enhancement, in which Au NPs serve as the nuclei for precipitation of a Ag coating, the thickness of which can be easily controlled by varying the ratio of the reactants. After synthesis, electron microscopy showed that the Au-Ag NPs displayed a core-shell structure, and that they could be successfully immobilized onto a cellulose membrane by heat treatment. We then investigated the antibacterial properties of this NP-coated cellulose paper against E. coli JM109. The inhibition rate, growth curve, and AATCC 100 activity test showed that cellulose paper coated with 15 nm Au-Ag NPs possessed excellent antibacterial activity against E. coli JM109. These results suggest that Au-Ag NPs immobilized on cellulose paper could be a valuable antibacterial technology for applications such as food packaging, clothing, wound dressings, and other personal care products.

In this work, we investigate highly sensitive fluorescent Cu nanoparticles for use as rapid and specific nucleic acid amplification nanoprobes (NPs) for the diagnosis of tuberculosis. After applying polymerase chain reaction (PCR) to a tuberculosis (TB) sample, we demonstrate that the presence of the targeted IS6110 DNA sequence of TB can be easily and directly detected through the in situ formation of DNA-templated fluorescent Cu NPs and subsequently quantified using only a smartphone. Compared to traditional DNA analysis, this sensing platform does not require purification steps and eliminates the need for electrophoresis to confirm the PCR results. After optimization, this dsDNA-Cu NP-PCR method has the ability to analyze clinical TB nucleic acid samples at a detection limit of 5 fg/μL, and the fluorescent signal can be distinguished in only ∼3 min after the DNA has been amplified. Moreover, with the combination of smartphone-assisted imaging analysis, we can further reduce the instrument size/cost and enhance the portability. In this manner, we are able to eliminate the need for a fluorescent spectrophotometer to measure the clinical sample. These results demonstrate this platform's practical applicability, combining a smartphone and on-site analysis while retaining the detection performance, making it suitable for clinical DNA applications in resource-limited regions of the world.

The diagnosis of periprosthetic joint infection (PJI) remains a challenge. However, recent studies showed that synovial fluid biomarkers have demonstrated greater diagnostic accuracy than the currently used PJI diagnostic tests. In many diagnostic tests, combining several biomarkers into panels is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases, and reducing cost. In this study, we prove that combining alpha-defensin and C-reactive protein (CRP) as biomarkers possesses the potential to provide accurate PJI diagnosis. To further verify the result, we developed a multi-target lateral flow immunoassay strip (msLFIA) with staking pad design to obtain on-site rapid response for clinical diagnosis of PJI. A total of 10 synovial fluid samples were tested using the msLFIA, and the results showed that the combined measurements of synovial fluid alpha-defensin and CRP levels were consistent with those obtained from a commercial enzyme-linked immunosorbent assay kit. In addition, we developed a multi-target lateral flow immunoassay strip (msLFIA) with staking pad design to obtain on-site rapid response for clinical diagnosis of PJI, which the multi-target design is used to increase specificity and the stacking pad design is to enhance detection sensitivity. As a result, the turnaround time of the highly sensitive test can be limited from several hours to 20 min. We expect that the developed msLFIA possesses the potential for routine monitoring of PJI as a convenient, low-cost, rapid and easy to use detection device for PJI.

Sulfatase 1 (SULF1) plays a key role in cell signaling involving in cell growth, differentiation, proliferation, and migration. Abnormal SULF1 expression has been implicated in the development of various cancers and diseases of the skeletal and nervous systems. The present study aims to examine the difference in SULF1 expression between degenerative and non-degenerative intervertebral discs (IVDs) to provide an enhanced understanding of disc degeneration. Degenerative and non-degenerative disc tissues were surgically harvested from patients and experimental rats. Disc degeneration-specific genes were identified by microarray analysis. The gene expression of SULF1 was measured by sulfatase assay, reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blotting. Also, the presence of SULF1 in human and rat discs was confirmed by immunohistochemistry. More specifically in human cells, an increase of SULF1 gene expression was observed in degenerative cells at both mRNA and protein levels, as well as in time- and dose-dependent manner in response to TNF-α treatment. Increased staining of SULF1 was detected in degenerative discs compared to non-degenerative discs for humans and rats. These findings show an upregulation of SULF1 in degenerative discs for the first time, and suggest that there is a link between SULF1 and disc degeneration.

Postoperative symptomatic epidural hematoma (SEH) is a serious complication of lumbar spine surgery. Despite its rarity, this uncommon complication may result in devastating neurological sequelae, including lower limb weakness. A retrospective study was made to identify possible risk factors of postoperative spinal epidural hematoma by reviewing the clinical cases of this rare complication and analyzing the postoperative evaluations of patients. From 2002 to 2010, out of 15,562 who underwent lumbar decompression procedure with/without instrumentation, 25 patients required reoperation for epidural hematoma after the initial spinal surgery. For the control group, another 75 patients were randomly selected from the pool of patients who received lumbar decompression surgery during the same period of time. The medical records of preoperative, intraoperative and postoperative factors were collected to determine possible risk factors by comparing between the cases and controls, and the postoperative evaluations of muscle power, intractable pain, saddle anesthesia, time to detection and time to evacuation were analyzed to find if there is any significant relation within the case group. Mann–Whitney U test, two-sample t test, χ 2 test and Fisher’s exact test were used for statistical analysis. The incidence of postoperative symptomatic epidural hematoma is 0.16 %. After the initial procedure, 20 (80 %) patients developed progressive decrease in muscle power (MP ≤ 3), 14 (56 %) patients had intractable pain (VAS ≥ 7), and 19 (76 %) patients had saddle anesthesia. Preoperative diastolic blood pressure, intraoperative use of gelfoam for dura coverage and postoperative drain output were statistically significant risk factors (p

Diabetes mellitus (DM) has been clinically proved as a risk factor of disc degeneration, and the accumulation of advanced glycation end products (AGEs) is known to be potentially involved in diabetes. The purpose of this study is to investigate the effect of AGEs in the degeneration process of diabetic nucleus pulposus (NP) in rats and humans. Diabetic NP cells from rat coccygeal discs were treated with different concentrations of AGEs (0, 50, and 100 µg/ml) for 3 days, and mRNA expressions of MMP-2 and RAGE were measured by real-time RT-PCR. In addition, conditioned medium from NP cells was used to analyze protein expression of MMP-2 activity and ERK by gelatin zymography and Western blot. These experiments were repeated using human intervertebral disc samples. The immunohistochemical expression of AGEs was significantly increased in diabetic discs. In response to AGEs, an increase of MMP-2, RAGE, and ERK at both mRNA and protein expression levels was observed in diabetic NP cells. The findings suggest that AGEs and DM are associated with disc degeneration in both species. Hyperglycemia in diabetes enhances the accumulation of AGEs in the NP and triggers disc degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:238–244, 2014.

Sulfatase 1 (SULF1) plays a key role in cell signaling involving in cell growth, differentiation, proliferation, and migration. Abnormal SULF1 expression has been implicated in the development of various cancers and diseases of the skeletal and nervous systems. The present study aims to examine the difference in SULF1 expression between degenerative and non-degenerative intervertebral discs (IVDs) to provide an enhanced understanding of disc degeneration. Degenerative and non-degenerative disc tissues were surgically harvested from patients and experimental rats. Disc degeneration-specific genes were identified by microarray analysis. The gene expression of SULF1 was measured by sulfatase assay, reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blotting. Also, the presence of SULF1 in human and rat discs was confirmed by immunohistochemistry. More specifically in human cells, an increase of SULF1 gene expression was observed in degenerative cells at both mRNA and protein levels, as well as in time- and dose-dependent manner in response to TNF-α treatment. Increased staining of SULF1 was detected in degenerative discs compared to non-degenerative discs for humans and rats. These findings show an upregulation of SULF1 in degenerative discs for the first time, and suggest that there is a link between SULF1 and disc degeneration.

Diabetes mellitus (DM) has been clinically proved as a risk factor of disc degeneration, and the accumulation of advanced glycation end products (AGEs) is known to be potentially involved in diabetes. The purpose of this study is to investigate the effect of AGEs in the degeneration process of diabetic nucleus pulposus (NP) in rats and humans. Diabetic NP cells from rat coccygeal discs were treated with different concentrations of AGEs (0, 50, and 100 µg/ml) for 3 days, and mRNA expressions of MMP-2 and RAGE were measured by real-time RT-PCR. In addition, conditioned medium from NP cells was used to analyze protein expression of MMP-2 activity and ERK by gelatin zymography and Western blot. These experiments were repeated using human intervertebral disc samples. The immunohistochemical expression of AGEs was significantly increased in diabetic discs. In response to AGEs, an increase of MMP-2, RAGE, and ERK at both mRNA and protein expression levels was observed in diabetic NP cells. The findings suggest that AGEs and DM are associated with disc degeneration in both species. Hyperglycemia in diabetes enhances the accumulation of AGEs in the NP and triggers disc degeneration.