Surface-enhanced Raman Scattering Microneedle For Biological Detection Study

Recently,microneedles-based biological detection has shown extensive promise for the minimally invasive and painless diagnosis and treatment of diseases.It mainly includes the chemical sensing of biomarkers in skin interstitial fluid（ISF）and sensing of its own physical or chemical parameters,which has two major challenges:（1）complex and time-consuming detection procedures,usually involving in vivo ISF extraction and in vitro ISF release;（2）limited variety of biomarkers detectable in ISF due to the limitations of in situ sensing technologies.Hence,developing new in situ sensing technologies with high sensitivity and reliability is the key to expanding microneedle sensing applications.In this thesis,new surface-enhanced Raman scattering（SERS）microneedles are designed and their bio-detection capability is investigated,enabling microneedle technology for auxiliary disease diagnostics.The main contents are outlined below:1.Development of internal standardized SERS microneedles with core-satellite gold nanoparticles deposition.An internal standardized surface-enhanced Raman scattering microneedle（IS-SERS-MN）is developed,which mainly consists of a polymer PMMA microneedle substrate,core-satellite gold nanoparticles,and an internal standard（4-MBN）adsorbed on the gold core.The core-satellite gold nanoparticles are equipped on the microneedle surface by electrostatic adsorption of amphiphilic polymer（PS-b-P4VP）and in situ gold growth.PS-b-P4VP not only ensures the density of adsorbed gold nanoparticles on the microneedle surface but also induces a monodisperse state of gold nanoparticles,which improves the reproducibility of IS-SERS-MN analysis.Importantly,the introduction of the internal standard can correct errors due to changes in detection conditions,etc.,which further improves the accuracy of IS-SERS-MN analysis.The enhancement factor（EF）of the developed IS-SERS-MN is 9.49?10⁴ and the high EF provides the basis for direct sensing of analytes in the ISF,e.g.,CV of 0.10 n M can be detected.It is shown that IS-SERS-MN has good in vivo application,including effective penetration of the skin stratum corneum to reach the dermal ISF,rapid recovery after insertion into the skin,high stability of gold nanoparticles adsorbed on the microneedles before and after skin insertion,etc.This study expands the sensing technique of microneedles,and the developed IS-SERS-MN can be used in various sensing related to ISF.In addition,the proposed SERS microneedle fabrication method is versatile.And new SERS microneedles for different types can be designed by changing the type or morphology of nanomaterials,polymer microneedle materials.2.Direct sensing of bacterial metabolites by SERS microneedles for infection diagnosis.Clinical diagnosis of bacterial infections often relies on colony culture methods,which has long test times and are difficult to meet the needs for point-of-care of diagnosis.Bacterial metabolites have been found to be transferred into biological fluids as biomarkers for infection.But the possibilities and potential of using bacterial metabolites in ISF for infection diagnosis have not been fully explored.By using pyocyanine as a representative bacterial metabolite,sensitive and reliable detection of bacterial metabolites in dermal ISF is achieved via the IS-SERS-MN.The sensing proformances of IS-SERS-MN for pyocyanine in ISF,including detection speed,sensitivity and working curve,are investigated in an in vitro ISF model.The sensing feasibility and accuracy of quantifying of pyocyanine in the skin ISF of living mice are demonstrated.IS-SERS-MN is also used to reveal whether pyocyanine in subcutaneous tissues can be transferred to the dermal ISF.Bacterial infection diagnosis in a real mouse wound indicates the feasibility of the IS-SERS-MN method.The results show that IS-SERS-MN can detect pyocyanine in ISF down to 0.30μM,which is about two orders of magnitude lower than the concentration of pyocyanine in clinically reported biological samples.LC-MS results indicate that IS-SERS-MN has reliable quantification capability for pyocyanine in the ISF of living mice.In addition,IS-SERS-MN can detect the Raman signal of pyocyanine secreted by pseudomonas aeruginosa in the actual wound infection site,and the signal changes at different times are highly correlated with the changes of the bacterial count at the wound.This study provides a new route for rapid,minimally-invasive,and painless diagnosis of bacterial infections,and is expected to promote the clinical application of microneedle technology.3.Indirect sensing of H₂O₂ by SERS microneedles for tracing the development of peritonitis.Real-time dynamic tracking of acute peritonitis can effectively reduce acute reactions such as decreased blood pressure and systemic toxicity caused by emergencies.Blood testing faces the risk of patient pain and disease transmission.The diagnostic modalities such as peritoneal CT and X-ray have drawbacks such as cumbersome processes and radiation damage.In this study,the development of acute peritonitis is dynamically tracked by detecting H₂O₂ in peritoneal skin ISF via SERS tags labeled microneedle technique.SERS tags consist mainly of core-satellite gold nanoparticles and 3-mercaptophenylboronic acid.Since H₂O₂ cannot be directly responded by the core-satellite gold nanoparticles on the microneedle,this study uses the oxidation of 3-mercaptophenylboronic acid and the appearance of new Raman peaks to detect H₂O₂ indirectly.The results show that the developed SERS tags labeled microneedle has high sensitivity and selectivity for H₂O₂ sensing and can reliably quantify the H₂O₂ content in ISF.The results of an acute peritonitis model in mice show that the Raman signal changes of H₂O₂ in ISF are consistent with the trend of inflammatory factor concentration in plasma.The Raman signal changes of H₂O₂ in the ISF differ under different drug interventions and are consistent with histopathological results in the peritoneum.These results suggest that the developed SERS tags labeled microneedle technique can dynamically track the progression of peritonitis and evaluate the therapeutic effects of different drugs.4.Near-infrared light-responsive SERS for revealing the local temperature of microneedles in vivo.Stimulus-responsive microneedle technology enables on-demand drug delivery to skin ISF or in vivo tissues and shows great potential in the field of minimally invasive therapy for diseases.Among them,near-infrared（NIR）light is an effective external stimulus in vivo applications（e.g.controlled-release drugs,photothermal therapy）,due to its high penetration capacity.The increase in local temperature around the in vivo microneedles after absorption of NIR light irradiation is a prerequisite for on-demand drug delivery or photothermal therapy with microneedles in the tissues,but excessive local temperature can damage adjacent tissues.Therefore,development of local temperature sensing methods is useful to the safe and effective application of NIR light-responsive microneedle technology in vivo.In this study,a NIR light-responsive surface-enhanced Raman scattering technique is developed to measure the local temperature of microneedles in vivo.The key to sensing is the elaborately screening of Raman dyes（DTTC）as reporters that are highly sensitive to NIR light irradiation.It is found that flexible polymethyl chains and strong absorption at NIR light region are two essential conditions for the selection of reporter molecules.As a proof of concept,IS-SERS-MN is still chosen as the test model.Under the NIR light irradiation,not only the core-satellite gold nanoparticles can generate heat to enhance the local temperature of microneedles,but also the DTTC bound to the core-satellite gold nanoparticles will undergo photodegradation,leading to the reduction of their SERS signals.The results show that the SERS signal change of microneedles has a good linear relationship with the local temperature.After blocking the microneedles by pork tissue,the local temperature sensed by the SERS signal is consistent with the thermal imager results.Finally,the feasibility of the NIR light responsive SERS technique is verified in living tumor tissues.The developed NIR light-responsive SERS technique is expected to facilitate the widespread application of stimulus-responsive microneedles in vivo.